Polynucleotides encoding antigenic hiv type b polypeptides, polypeptides, and uses thereof

ABSTRACT

The present invention relates to polynucleotides encoding immunogenic HIV polypeptides. Uses of the polynucleotides in applications including immunization, generation of packaging cell lines, and production of HIV polypeptides are also described. Polynucleotides encoding antigenic HIV polypeptides are described, as are uses of these polynucleotides and polypeptide products therefrom, including formulations of immunogenic compositions and uses thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of Ser. No. 10/976,619, which is acontinuation-in-part of U.S. Ser. No. 10/190,434, filed Jul. 5, 2002which claims the benefit of U.S. Provisional Patent Applications Ser.No. 60/349,728, filed 16 Jan. 2002, and 60/316,860, filed 31 Aug. 2001,from which priority is claimed under 35 USC § 119(e)(1), and whichapplications are incorporated herein by reference in their entireties.

GOVERNMENT SUPPORT

The invention was supported, in whole or in part, by NIH HIVDDT GrantNo. N01-AI-05396 from the National Institutes of Health. The U.S.Government may have certain rights in the invention.

TECHNICAL FIELD

Polynucleotides encoding antigenic HIV polypeptides (e.g., those shownin Table C) are described, as are uses of these polynucleotides andpolypeptide products including formulations of immunogenic compositionsand uses thereof.

BACKGROUND OF THE INVENTION

Acquired immune deficiency syndrome (AIDS) is recognized as one of thegreatest health threats facing modern medicine. There is, as yet, nocure for this disease.

In 1983-1984, three groups independently identified the suspectedetiological agent of AIDS. See, e.g., Barre-Sinoussi et al. (1983)Science 220:868-871; Montagnier et al., in Human T-Cell Leukemia Viruses(Gallo, Essex & Gross, eds., 1984); Vilmer et al. (1984) (1984) TheLancet 1:753; Popovic et al. (1984) Science 224:497-500; Levy et al.(1984) Science 225:840-842. These isolates were variously calledlymphadenopathy-associated virus (LAV), human T-cell lymphotropic virustype III (HTLV-M), or AIDS-associated retrovirus (ARV). All of theseisolates are strains of the same virus, and were later collectivelynamed Human Immunodeficiency Virus (HIV). With the isolation of arelated AIDS-causing virus, the strains originally called HIV are nowtermed HIV-1 and the related virus is called HIV-2 See, e.g., Guyader etal. (1987) Nature 326:662-669; Brun-Vezinet et al. (1986) Science233:343-346; Clavel et al. (1986) Nature 324:691-695.

A great deal of information has been gathered about the HIV virus,however, to date an effective vaccine has not been identified. Severaltargets for vaccine development have been examined including the env andGag gene products encoded by HIV. Gag gene products include, but are notlimited to, Gag-polymerase and Gag-protease. Env gene products include,but are not limited to, monomeric gp120 polypeptides, oligomeric gp140polypeptides and gp160 polypeptides.

Haas, et al., (Current Biology 6(3):315-324, 1996) suggested thatselective codon usage by HIV-1 appeared to account for a substantialfraction of the inefficiency of viral protein synthesis. Andre, et al.,(J. Virol. 72(2):1497-1503, 1998) described an increased immune responseelicited by DNA vaccination employing a synthetic gp120 sequence withmodified codon usage. Schneider, et al., (J. Virol. 71(7):4892-4903,1997) discuss inactivation of inhibitory (or instability) elements (INS)located within the coding sequences of the Gag and Gag-protease codingsequences.

The Gag proteins of HIV-1 are necessary for the assembly of virus-likeparticles. HIV-1 Gag proteins are involved in many stages of the lifecycle of the virus including, assembly, virion maturation after particlerelease, and early post-entry steps in virus replication. The roles ofHIV-1 Gag proteins are numerous and complex (Freed, E. O., Virology251:1-15, 1998).

Wolf, et al., (PCT International Application, WO 96/30523, published 3Oct. 1996; European Patent Application, Publication No. 0 449 116 A1,published 2 Oct. 1991) have described the use of altered pr55 Gag ofHIV-1 to act as a non-infectious retroviral-like particulate carrier, inparticular, for the presentation of immunologically important epitopes.Wang, et al., (Virology 200:524-534, 1994) describe a system to studyassembly of HIV Gag-β-galactosidase fusion proteins into virions. Theydescribe the construction of sequences encoding HIV Gag-β-galactosidasefusion proteins, the expression of such sequences in the presence of HIVGag proteins, and assembly of these proteins into virus particles.

Shiver, et al., (PCT International Application, WO 98/34640, published13 Aug. 1998) described altering HIV-1 (CAM1) Gag coding sequences toproduce synthetic DNA molecules encoding HIV Gag and modifications ofHIV Gag. The codons of the synthetic molecules were codons preferred bya projected host cell.

Recently, use of HIV Env polypeptides in immunogenic compositions hasbeen described. (see, U.S. Pat. No. 5,846,546 to Hurwitz et al., issuedDec. 8, 1998, describing immunogenic compositions comprising a mixtureof at least four different recombinant virus that each express adifferent HIV env variant; and U.S. Pat. No. 5,840,313 to Vahlne et al.,issued Nov. 24, 1998, describing peptides which correspond to epitopesof the HIV-1 gp120 protein). In addition, U.S. Pat. No. 5,876,731 to Siaet al, issued Mar. 2, 1999 describes candidate vaccines against HIVcomprising an amino acid sequence of a T-cell epitope of Gag linkeddirectly to an amino acid sequence of a B-cell epitope of the V3 loopprotein of an HIV-1 isolate containing the sequence GPGR.

SUMMARY OF THE INVENTION

Described herein are novel HIV sequences, polypeptides encoded by thesenovel sequences, and synthetic expression cassettes generated from theseand other HIV sequences. In one aspect, the present invention relates toimproved HIV expression cassettes. In a second aspect, the presentinvention relates to generating an immune response in a subject usingthe expression cassettes of the present invention. In a further aspect,the present invention relates to generating an immune response in asubject using the expression cassettes of the present invention, as wellas, polypeptides encoded by the expression cassettes of the presentinvention. In another aspect, the present invention relates to enhancedvaccine technologies for the induction of potent neutralizing antibodiesand/or cellular immune responses against HIV in a subject.

In certain embodiments, the present invention relates to syntheticpolynucleotides and/or expression cassettes encoding HIV polypeptides,including, but not limited to, Env, Gag, Pol, RT, Int, Prot, Vpr, Vpu,Vif, Nef, Tat, Rev and/or fragments or combinations thereof. Inaddition, the present invention also relates to improved expression ofHIV polypeptides and production of virus-like particles. Syntheticexpression cassettes encoding the HIV polypeptides (e.g., Gag-, pol-,protease (prot)-, reverse transcriptase, integrase, RNAseH, Tat, Rev,Nef, Vpr, Vpu, Vif and/or Env-containing polypeptides) are described, asare uses of the expression cassettes. Mutations in some of the genes aredescribed that reduce or eliminate the activity of the gene productwithout adversely affecting the ability of the gene product to generatean immune response. Exemplary synthetic polynucleotides include, but arenot limited to, GagComplPolmut.SF2 (SEQ ID NO:9), GagComplPolmutAtt.SF2(SEQ ID NO:10), GagComplPolmutIna.SF2 (SEQ ID NO:11),gagCpolInaTatRevNef.opt_B (SEQ ID NO:12), GagPolmutAtt.SF2 (SEQ IDNO:13), GagPolmutIna.SF2 (SEQ ID NO:14), GagProtInaRTmut.SF2 (SEQ IDNO:15), GagProtInaRTmutTatRevNef.opt_B (SEQ ID NO:16), GagRTmut.SF2 (SEQID NO:17), GagTatRevNef.opt_B (SEQ ID NO:18), gp140.modSF162.CwtLmod(SEQ ID NO:19), gp 140.modSF162.CwtLnat (SEQ ID NO:20),gp160.modSF162.delV2.mut7 (SEQ ID NO:21), gp160.modSF162.delV2.mut8 (SEQID NO:22), int.opt.mut.SF2 (SEQ ID NO:23), int.opt.SF2 (SEQ ID NO:24),nef.D125G.-myr.opt.SF162 (SEQ ID NO:25), nef.D107G.-myr18.opt.SF162 (SEQID NO:26), nef.opt.D125G.SF162 (SEQ ID NO:27), nef.opt.SF162 (SEQ IDNO:28), p15RnaseH.opt.SF2 (SEQ ID NO:29), p2Pol.opt.YMWM.SF2 (SEQ IDNO:30), p2PolInaopt.YM.SF2 (SEQ ID NO:31), p2Polopt.SF2 (SEQ ID NO:32),p2PolTatRevNef.opt.native_B (SEQ ID NO:33), p2PolTatRevNef.opt_B (SEQ IDNO:34), pol.opt.SF2 (SEQ ID NO:35), prot.opt.SF2 (SEQ ID NO:36),protIna.opt.SF2 (SEQ ID NO:37), protInaRT.YM.opt.SF2 (SEQ ID NO:38),protInaRT.YMWM.opt.SF2 (SEQ ID NO:39), ProtInaRTmut.SF2 (SEQ ID NO:40),protRT.opt.SF2 (SEQ ID NO:41), ProtRT.TatRevNef.opt_B (SEQ ID NO:42),ProtRTTatRevNef.opt_B (SEQ ID NO:43), rev.exon1_(—)2.M5-10.opt.SF162(SEQ ID NO:44), rev.exon1_(—)2.opt.SF162 (SEQ ID NO:45), RT.opt.SF2(mutant) (SEQ ID NO:46), RT.opt.SF2 (native) (SEQ ID NO:47), RTmut.SF2(SEQ ID NO:48), tat.exon1_(—)2.opt.C22-37.5F2 (SEQ ID NO:49),tat.exon1_(—)2.opt.C37.5F2 (SEQ ID NO:50), TatRevNef.opt.native.SF162(SEQ ID NO:51), TatRevNef.opt.SF162 (SEQ ID NO:52), TatRevNefGag B (SEQID NO:53), TatRevNefgagCpollna B (SEQ ID NO:54),TatRevNefGagProtInaRTmut B (SEQ ID NO:55), TatRevNefp2Pol.opt_B, (SEQ IDNO:56) TatRevNefprotRTopt B (SEQ ID NO:57), vif.opt.SF2 (SEQ ID NO:58),vpr.opt.SF2 (SEQ ID NO:59), and vpu.opt.SF162 (SEQ ID NO:60).

Thus, one aspect of the present invention relates to expressioncassettes and polynucleotides contained therein. The expressioncassettes typically include an HIV-polypeptide encoding sequenceinserted into an expression vector backbone. In one embodiment, anexpression cassette comprises a polynucleotide sequence encoding one ormore polypeptides, wherein the polynucleotide sequence comprises asequence having between about 85% to 100% and any integer valuestherebetween, for example, at least about 85%, preferably about 90%,more preferably about 95%, and more preferably about 98% sequenceidentity to the sequences taught in the present specification.

The polynucleotides encoding the HIV polypeptides of the presentinvention may also include sequences encoding additional polypeptides.Such additional polynucleotides encoding polypeptides may include, forexample, coding sequences for other viral proteins (e.g., hepatitis B orC or other HIV proteins, such as, polynucleotide sequences encoding anHIV Gag polypeptide, polynucleotide sequences encoding an HIV Envpolypeptide and/or polynucleotides encoding one or more of vif, vpr,tat, rev, vpu and nef); cytokines or other transgenes.

In one embodiment, the sequence encoding the HIV Pol polypeptide(s) canbe modified by deletions of coding regions corresponding to reversetranscriptase and integrase. Such deletions in the polymerasepolypeptide can also be made such that the polynucleotide sequencepreserves T-helper cell and CTL epitopes. Other antigens of interest maybe inserted into the polymerase as well.

In another embodiment, an expression cassette comprises a polynucleotidesequence encoding a polypeptide, for example, GagComplPolmut.SF2 (SEQ IDNO:9), GagComplPolmutAtt.SF2 (SEQ ID NO:10), GagComplPolmutIna.SF2 (SEQID NO:11), gagCpolInaTatRevNef.opt_B (SEQ ID NO:12), GagPolmutAtt.SF2(SEQ ID NO:13), GagPolmutIna.SF2 (SEQ ID NO:14), GagProtInaRTmut.SF2(SEQ ID NO:15), GagProtInaRTmutTatRevNef.opt_B (SEQ ID NO:16),GagRTmut.SF2, (SEQ ID NO:17) GagTatRevNef.opt_B (SEQ ID NO:18),gp140.modSF162.CwtLmod (SEQ ID NO:19), gp140.modSF162.CwtLnat (SEQ IDNO:20), gp160.modSF162.delV2.mut7 (SEQ ID NO:21),gp160.modSF162.deIV2.mut8 (SEQ ID NO:22), int.opt.mut.SF2 (SEQ IDNO:23), int.opt.SF2 (SEQ ID NO:24), nef.D125G.-myr.opt.SF162 (SEQ IDNO:25), nef.D107G.-myr18.opt.SF162 (SEQ ID NO:26), nef.opt.D125G.SF162(SEQ ID NO:27), nef.opt.SF162 (SEQ ID NO:28), p15RnaseH.opt.SF2 (SEQ IDNO:29), p2Pol.opt.YMWM.SF2 (SEQ ID NO:30), p2PolInaopt.YM.SF2, (SEQ IDNO:31) p2Polopt.SF2 (SEQ ID NO:32), p2PolTatRevNef.opt.native_B (SEQ IDNO:33), p2PolTatRevNef.opt_B (SEQ ID NO:34), pol.opt.SF2 (SEQ ID NO:35),prot.opt.SF2 (SEQ ID NO:36), protIna.opt.SF2 (SEQ ID NO:37),protInaRT.YM.opt.SF2 (SEQ ID NO:38), protInaRT.YMWM.opt.SF2 (SEQ IDNO:39), ProtInaRTmut.SF2 (SEQ ID NO:40), protRT.opt.SF2 (SEQ ID NO:41),ProtRT.TatRevNef.opt_B (SEQ ID NO:42), ProtRTTatRevNef.opt_B (SEQ IDNO:43), rev.exon1_(—)2.M5-10.opt.SF162 (SEQ ID NO:44),rev.exon1_(—)2.opt.SF162 (SEQ ID NO:45), RT.opt.SF2 (mutant) (SEQ IDNO:46), RT.opt.SF2 (native) (SEQ ID NO:47), RTmut.SF2 (SEQ ID NO:48),tat.exon1_(—)2.opt.C22-37.5F2 (SEQ ID NO:49), tat.exon1_(—)2.opt.C37.5F2(SEQ ID NO:50), TatRevNef.opt.native.SF162 (SEQ ID NO:51),TatRevNef.opt.SF162 (SEQ ID NO:52), TatRevNefGag B (SEQ ID NO:53),TatRevNefgagCpollna B (SEQ ID NO:54), TatRevNefGagProtInaRTmut B (SEQ IDNO:55), TatRevNefp2Pol.opt_B (SEQ ID NO:56), TatRevNefprotRTopt B (SEQID NO:57), vif.opt.SF2 (SEQ ID NO:58), vpr.opt.SF2 (SEQ ID NO:59), andvpu.opt.SF162 (SEQ ID NO:60), wherein the polynucleotide sequenceencoding the polypeptide comprises a sequence having between about 85%to 100% and any integer values therebetween, for example, at least about85%, preferably about 90%, more preferably about 95%, and mostpreferably about 98% sequence identity to the sequences taught in thepresent specification.

The native and synthetic polynucleotide sequences encoding the HIVpolypeptides of the present invention typically have between about 85%to 100% and any integer values therebetween, for example, at least about85%, preferably about 90%, more preferably about 95%, and morepreferably about 98% sequence identity to the sequences taught herein.Further, in certain embodiments, the polynucleotide sequences encodingthe HIV polypeptides of the invention will exhibit 100% sequenceidentity to the sequences taught herein.

The polynucleotides of the present invention can be produced byrecombinant techniques, synthetic techniques, or combinations thereof.

The present invention further includes recombinant expression systemsfor use in selected host cells, wherein the recombinant expressionsystems employ one or more of the polynucleotides and expressioncassettes of the present invention. In such systems, the polynucleotidesequences are operably linked to control elements compatible withexpression in the selected host cell. Numerous expression controlelements are known to those in the art, including, but not limited to,the following: transcription promoters, transcription enhancer elements,transcription termination signals, polyadenylation sequences, sequencesfor optimization of initiation of translation, and translationtermination sequences. Exemplary transcription promoters include, butare not limited to those derived from CMV, CMV+intron A, SV40, RSV,HIV-Ltr, MMLV-ltr, and metallothionein.

In another aspect the invention includes cells comprising one or more ofthe expression cassettes of the present invention where thepolynucleotide sequences are operably linked to control elementscompatible with expression in the selected cell. In one embodiment suchcells are mammalian cells. Exemplary mammalian cells include, but arenot limited to, BHK, VERO, HT1080, 293, RD, COS-7, and CHO cells. Othercells, cell types, tissue types, etc., that may be useful in thepractice of the present invention include, but are not limited to, thoseobtained from the following: insects (e.g., Trichoplusia ni (Tn5) andSf9), bacteria, yeast, plants, antigen presenting cells (e.g.,macrophage, monocytes, dendritic cells, B-cells, T-cells, stem cells,and progenitor cells thereof), primary cells, immortalized cells,tumor-derived cells.

In a further aspect, the present invention includes compositions forgenerating an immunological response, where the composition typicallycomprises at least one of the expression cassettes of the presentinvention and may, for example, contain combinations of expressioncassettes such as one or more expression cassettes carrying aPol-derived-polypeptide-encoding polynucleotide, one or more expressioncassettes carrying a Gag-derived-polypeptide-encoding polynucleotide,one or more expression cassettes carrying accessory polypeptide-encodingpolynucleotides (e.g., native or synthetic vpu, vpr, nef, vif, tat,rev), and/or one or more expression cassettes carrying anEnv-derived-polypeptide-encoding polynucleotide. Such compositions mayfurther contain an adjuvant or adjuvants. The compositions may alsocontain one or more HIV polypeptides. The HIV polypeptides maycorrespond to the polypeptides encoded by the expression cassette(s) inthe composition, or may be different from those encoded by theexpression cassettes. In compositions containing both expressioncassettes (or polynucleotides of the present invention) andpolypeptides, various expression cassettes of the present invention canbe mixed and/or matched with various HIV polypeptides described herein.

In another aspect the present invention includes methods of immunizationof a subject. In the method any of the above described compositions areinto the subject under conditions that are compatible with expression ofthe expression cassette(s) in the subject. In one embodiment, theexpression cassettes (or polynucleotides of the present invention) canbe introduced using a gene delivery vector. The gene delivery vectorcan, for example, be a non-viral vector or a viral vector. Exemplaryviral vectors include, but are not limited to eucaryotic layered vectorinitiation systems, Sindbis-virus (or other alphavirus) derived vectors,retroviral vectors, and lentiviral vectors. Other exemplary vectorsinclude, but are not limited to, pCMVKm2, pCMV6a, pCMV-link, andpCMVPLEdhfr. Compositions useful for generating an immunologicalresponse can also be delivered using a particulate carrier (e.g., PLG orCTAB-PLG microparticles). Further, such compositions can be coated on,for example, gold or tungsten particles and the coated particlesdelivered to the subject using, for example, a gene gun. Thecompositions can also be formulated as liposomes. In one embodiment ofthis method, the subject is a mammal and can, for example, be a human.

In a further aspect, the invention includes methods of generating animmune response in a subject. Any of the expression cassettes describedherein can be expressed in a suitable cell to provide for the expressionof the HIV polypeptides encoded by the polynucleotides of the presentinvention. The polypeptide(s) are then isolated (e.g., substantiallypurified) and administered to the subject in an amount sufficient toelicit an immune response. In certain embodiments, the methods compriseadministration of one or more of the expression cassettes orpolynucleotides of the present invention, using any of the gene deliverytechniques described herein. In other embodiments, the methods compriseco-administration of one or more of the expression cassettes orpolynucleotides of the present invention and one or more polypeptides,wherein the polypeptides can be expressed from these polynucleotides orcan be other HIV polypeptides. In other embodiments, the methodscomprise co-administration of multiple expression cassettes orpolynucleotides of the present invention. In still further embodiments,the methods comprise co-administration of multiple polypeptides, forexample polypeptides expressed from the polynucleotides of the presentinvention and/or other HIV polypeptides.

The invention further includes methods of generating an immune responsein a subject, where cells of a subject are transfected with any of theabove-described expression cassettes or polynucleotides of the presentinvention, under conditions that permit the expression of a selectedpolynucleotide and production of a polypeptide of interest (e.g.,encoded by any expression cassette of the present invention). By thismethod an immunological response to the polypeptide is elicited in thesubject. Transfection of the cells may be performed ex vivo and thetransfected cells are reintroduced into the subject. Alternately, or inaddition, the cells may be transfected in vivo in the subject. Theimmune response may be humoral and/or cell-mediated (cellular). In afurther embodiment, this method may also include administration of anHIV polypeptides before, concurrently with, and/or after introduction ofthe expression cassette into the subject.

The polynucleotides of the present invention may be employed singly orin combination. The polynucleotides of the present invention, encodingHIV-derived polypeptides, may be expressed in a variety of ways,including, but not limited to the following: a polynucleotide encoding asingle gene product (or portion thereof) expressed from a promoter;multiple polynucleotides encoding a more than one gene product (orportion thereof) (e.g., polycistronic coding sequences); multiplepolynucleotides in-frame to produce a single polyprotein; and, multiplepolynucleotides in-frame to produce a single polyprotein wherein thepolyprotein has protein cleavage sites between one or more of thepolypeptides comprising the polyprotein.

These and other embodiments of the present invention will readily occurto those of ordinary skill in the art in view of the disclosure herein.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A to 1D depict the nucleotide sequence of HIV Type C8_(—)5_TV1—C.ZA (SEQ ID NO:1; referred to herein as TV1). Variousregions are shown in Table A.

FIGS. 2A-C depicts an alignment of Env polypeptides from various HIVisolates (SF162, SEQ ID NO:2; TV1.8_(—)2, SEQ ID NO:3; TV1.8_(—)5, SEQID NO:4; TV2.12-5/1, SEQ ID NO:5; Consensus Sequence, SEQ ID NO:6). Theregions between the arrows indicate regions (of TV1 and TV2 clones, bothHIV Type C isolates) in the beta and/or bridging sheet region(s) thatcan be deleted and/or truncated. The “*” denotes N-linked glycosylationsites (of TV1 and TV2 clones), one or more of which can be modified(e.g., deleted and/or mutated).

FIG. 3 presents a schematic diagram showing the relationships betweenthe following forms of the HIV Env polypeptide: gp160, gp140, gp120, andgp41.

FIG. 4 presents exemplary data concerning transactivation activity ofTat mutants on LTR-CAT plasmid expression in 293 cells.

FIG. 5 presents exemplary data concerning export activity of Rev mutantsmonitored by CAT expression.

FIG. 6, sheets 1 and 2, presents the sequence of GagComplPolmut.SF2 (SEQID NO:9).

FIG. 7, sheets 1 and 2, presents the sequence of GagComplPolmutAtt.SF2(SEQ ID NO:10).

FIG. 8, sheets 1 and 2, presents the sequence of GagComplPolmutIna.SF2(SEQ ID NO:11).

FIG. 9, sheets 1 and 2, presents the sequence ofgagCpolInaTatRevNef.opt_B (SEQ ID NO: 12).

FIG. 10, sheets 1 and 2, presents the sequence of GagPolmutAtt.SF2 (SEQID NO:13).

FIG. 11, sheets 1 and 2, presents the sequence of GagPolmutIna.SF2 (SEQID NO: 14).

FIG. 12, sheets 1 and 2, presents the sequence of GagProtInaRTmut.SF2(SEQ ID NO:15).

FIG. 13, sheets 1 and 2, presents the sequence ofGagProtInaRTmutTatRevNef.opt_B (SEQ ID NO:16).

FIG. 14, sheets 1 and 2, presents the sequence of GagRTmut.SF2 (SEQ IDNO:17).

FIG. 15, presents the sequence of GagTatRevNef.opt_B (SEQ ID NO:18).

FIG. 16, presents the sequence of gp140.modSF162.CwtLmod (SEQ ID NO:19).

FIG. 17, presents the sequence of gp140.modSF162.CwtLnat (SEQ ID NO:20).

FIG. 18, presents the sequence of gp160.modSF162.delV2.mut7 (SEQ IDNO:21).

FIG. 19, presents the sequence of gp160.modSF162.delV2.mut8 (SEQ IDNO:22).

FIG. 20, presents the sequence of int.opt.mut.SF2 (SEQ ID NO:23).

FIG. 21, presents the sequence of int.opt.SF2 (SEQ ID NO:24).

FIG. 22, presents the sequence of nef.D125G.-myr.opt.SF162 (SEQ IDNO:25).

FIG. 23, presents the sequence of nef.D107G.-myr18.opt.SF162 (SEQ IDNO:26).

FIG. 24, presents the sequence of nef.opt.D125G.SF162 (SEQ ID NO:27).

FIG. 25, presents the sequence of nef.opt.SF162 (SEQ ID NO:28).

FIG. 26, presents the sequence of p15RnaseH.opt.SF2 (SEQ ID NO:29).

FIG. 27, presents the sequence of p2Pol.opt.YMWM.SF2 (SEQ ID NO:30).

FIG. 28, presents the sequence of p2PolInaopt.YM.SF2 (SEQ ID NO:31).

FIG. 29, presents the sequence of p2Polopt.SF2 (SEQ ID NO:32).

FIG. 30, presents the sequence of p2PolTatRevNef.opt.native_B (SEQ IDNO:33).

FIG. 31, sheets 1 and 2, presents the sequence of p2PolTatRevNef.opt_B(SEQ ID NO:34).

FIG. 32, presents the sequence of pol.opt.SF2 (SEQ ID NO:35).

FIG. 33, presents the sequence of prot.opt.SF2 (SEQ ID NO:36).

FIG. 34, presents the sequence of protIna.opt.SF2 (SEQ ID NO:37).

FIG. 35, presents the sequence of protInaRT.YM.opt.SF2 (SEQ ID NO:38).

FIG. 36, presents the sequence of protInaRT.YMWM.opt.SF2 (SEQ ID NO:39).

FIG. 37, presents the sequence of ProtInaRTmut.SF2 (SEQ ID NO:40).

FIG. 38, presents the sequence of protRT.opt.SF2 (SEQ ID NO:41).

FIG. 39, presents the sequence of ProtRT.TatRevNef.opt_B (SEQ ID NO:42).

FIG. 40, presents the sequence of ProtRTTatRevNef.opt_B (SEQ ID NO:43).

FIG. 41, presents the sequence of rev.exon1_(—)2.M5-10.opt.SF162 (SEQ IDNO:44).

FIG. 42, presents the sequence of rev.exon1_(—)2.opt.SF162 (SEQ IDNO:45).

FIG. 43, presents the sequence of RT.opt.SF2 (mutant) (SEQ ID NO:46).

FIG. 44, presents the sequence of RT.opt.SF2 (native) (SEQ ID NO:47).

FIG. 45, presents the sequence of RTmut.SF2 (SEQ ID NO:48).

FIG. 46, presents the sequence of tat.exon1_(—)2.opt.C22-37.5F2 (SEQ IDNO:49).

FIG. 47, presents the sequence of tat.exon1_(—)2.opt.C37.5F2 (SEQ IDNO:50).

FIG. 48, presents the sequence of TatRevNef.opt.native.SF162 (SEQ IDNO:51).

FIG. 49, presents the sequence of TatRevNef.opt.SF162 (SEQ ID NO:52).

FIG. 50, presents the sequence of TatRevNefGag B (SEQ ID NO:53).

FIG. 51, sheets 1 and 2, presents the sequence of TatRevNefgagCpolIna B(SEQ ID NO:54).

FIG. 52, sheets 1 and 2, presents the sequence ofTatRevNefGagProtInaRTmut B (SEQ ID NO:55).

FIG. 53, presents the sequence of TatRevNefp2Pol.opt_B (SEQ ID NO:56).

FIG. 54, presents the sequence of TatRevNefprotRTopt B (SEQ ID NO:57).

FIG. 55, presents the sequence of vif.opt.SF2 (SEQ ID NO:58).

FIG. 56, presents the sequence of vpr.opt.SF2 (SEQ ID NO:59).

FIG. 57, presents the sequence of vpu.opt.SF162 (SEQ ID NO:60).

FIG. 58, presents the sequence of gp140modSF162.GM135-154-186-195 (SEQID NO:61).

FIG. 59, presents the sequence of gp140modSF162.GM154 (SEQ ID NO:62).

FIG. 60, presents the sequence of gp140modSF162.GM154-186-195 (SEQ IDNO:63).

FIG. 61, presents the sequence of gp140mut7.modSF162.GM154 (SEQ IDNO:64).

FIG. 62 depicts alignment of amino acid sequences of various Envglycosylation mutants (GM), including amino acid translation ofgp140modSF162 (SEQ ID NO:65); translation of gp140.modSF162.GM154 (SEQID NO:66); translation of gp140.modSF162.GM154-186-195 (SEQ ID NO:67);and gp140.modSF162.GM135-154-186-195 (SEQ ID NO:68).

FIG. 63 presents an overview of genome organization of HIV-1 and usefulsubgenomic fragments.

FIG. 64 presents antibody titer data from immunized rabbits followingimmunization with HIV Envelope DNA constructs and protein.

FIG. 65 presents a comparison of ELISA titers against subtype B and CEnvelope proteins in rabbit sera collected after three DNA immunizationsand a single protein boost.

FIG. 66 presents data of neutralizing antibody responses against subtypeB SF162 EnvdV2 strain in rabbits immunized with subtype C TV1 Env in aDNA prime protein boost regimen.

FIG. 67 presents data of neutralizing antibody responses against subtypeC primary strains, TV1 and TV2 in 5.25 reporter cell assay after asingle protein boost.

FIG. 68 presents data of neutralizing antibody responses against subtypeC, TV1 and Du174, and subtype B, SF162 after a single protein boost (asmeasured by Duke PBMC assay).

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of chemistry, biochemistry, molecularbiology, immunology and pharmacology, within the skill of the art. Suchtechniques are explained fully in the literature. See, e.g., Remington'sPharmaceutical Sciences, 18th Edition (Easton, Pa.: Mack PublishingCompany, 1990); Methods In Enzymology (S. Colowick and N. Kaplan, eds.,Academic Press, Inc.); and Handbook of Experimental Immunology, Vols.I-IV (D. M. Weir and C. C. Blackwell, eds., 1986, Blackwell ScientificPublications); Sambrook, et al., Molecular Cloning: A Laboratory Manual(2nd Edition, 1989); Short Protocols in Molecular Biology, 4th ed.(Ausubel et al. eds., 1999, John Wiley & Sons); Molecular BiologyTechniques: An Intensive Laboratory Course, (Ream et al., eds., 1998,Academic Press); PCR (Introduction to Biotechniques Series), 2nd ed.(Newton & Graham eds., 1997, Springer Verlag).

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.

As used in this specification, the singular forms “a,” “an” and “the”include plural references unless the content clearly dictates otherwise.Thus, for example, reference to “an antigen” includes a mixture of twoor more such agents.

1. DEFINITIONS

In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below.

“Synthetic” sequences, as used herein, refers to HIVpolypeptide-encoding polynucleotides whose expression has been modifiedas described herein, for example, by codon substitution, alteredactivities, and/or inactivation of inhibitory sequences. “Wild-type” or“native” sequences, as used herein, refers to polypeptide encodingsequences that are essentially as they are found in nature, e.g., Gag,Pol, Vif, Vpr, Tat, Rev, Vpu, Env and/or Nef encoding sequences as foundin HIV isolates, e.g., SF162, SF2, AF110965, AF110967, AF110968,AF110975, 8_(—)5_TV1_C.ZA, 8_(—)2_TV1_C.ZA or 12-5_(—)1_TV2_C.ZA. Thevarious regions of the HIV genome are shown in Table A, with numberingrelative to 8_(—)5_TV1_C.ZA (FIGS. 1A-1D). Thus, the term “Pol” refersto one or more of the following polypeptides: polymerase (p6Pol);protease (prot); reverse transcriptase (p66RT or RT); RNAseH(p15RNAseH); and/or integrase (p31Int or Int). Identification of generegions for any selected HIV isolate can be performed by one of ordinaryskill in the art based on the teachings presented herein and theinformation known in the art, for example, by performing alignmentsrelative to 8_(—)5_TV1_C.ZA (FIGS. 1A-1D) or alignment to other knownHIV isolates, for example, Subtype B isolates with gene regions (e.g.,SF2, GenBank Accession number K02007; SF162, GenBank Accession NumberM38428, both herein incorporated by reference) and Subtype C isolateswith gene regions (e.g., GenBank Accession Number AF110965 and GenBankAccession Number AF110975, both herein incorporated by reference).

As used herein, the term “virus-like particle” or “VLP” refers to anonreplicating, viral shell, derived from any of several virusesdiscussed further below. VLPs are

generally composed of one or more viral proteins, such as, but notlimited to those proteins referred to as capsid, coat, shell, surfaceand/or envelope proteins, or particle-forming polypeptides derived fromthese proteins. VLPs can form spontaneously upon recombinant expressionof the protein in an appropriate expression system. Methods forproducing particular VLPs are known in the art and discussed more fullybelow. The presence of VLPs following recombinant expression of viralproteins can be detected using conventional techniques known in the art,such as by electron microscopy, X-ray crystallography, and the like.See, e.g., Baker et al., Biophys. J. (1991) 60:1445-1456; Hagensee etal., J. Virol. (1994) 68:4503-4505. For example, VLPs can be isolated bydensity gradient centrifugation and/or identified by characteristicdensity banding. Alternatively, cryoelectron microscopy can be performedon vitrified aqueous samples of the VLP preparation in question, andimages recorded under appropriate exposure conditions.

By “particle-forming polypeptide” derived from a particular viralprotein is meant a full-length or near full-length viral protein, aswell as a fragment thereof, or a viral protein with internal deletions,which has the ability to form VLPs under conditions that favor VLPformation. Accordingly, the polypeptide may comprise the full-lengthsequence, fragments, truncated and partial sequences, as well as analogsand precursor forms of the reference molecule. The term thereforeintends deletions, additions and substitutions to the sequence, so longas the polypeptide retains the ability to form a VLP. Thus, the termincludes natural variations of the specified polypeptide sincevariations in coat proteins often occur between viral isolates. The termalso includes deletions, additions and substitutions that do notnaturally occur in the reference protein, so long as the protein retainsthe ability to form a VLP. Preferred substitutions are those which areconservative in nature, i.e., those substitutions that take place withina family of amino acids that are related in their side chains.Specifically, amino acids are generally divided into four families: (1)acidic—aspartate and glutamate; (2) basic—lysine, arginine, histidine;(3) non-polar—alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan; and (4) uncharged polar—glycine,asparagine, glutamine, cystine, serine threonine, tyrosine.Phenylalanine, tryptophan, and tyrosine are sometimes classified asaromatic amino acids.

The term “HIV polypeptide” refers to any amino acid sequence thatexhibits sequence homology to native HIV polypeptides (e.g., Gag, Env,Prot, Pol, RT, Int, vif, vpr, vpu, tat, rev, nef and/or combinationsthereof) and/or which is functional. Non-limiting examples of functionsthat may be exhibited by HIV polypeptides include, use as immunogens(e.g., to generate a humoral and/or cellular immune response), use indiagnostics (e.g., bound by suitable antibodies for use in ELISAs orother immunoassays) and/or polypeptides which exhibit one or morebiological activities associated with the wild type or synthetic HIVpolypeptide. For example, as used herein, the term “Gag polypeptide” mayrefer to a polypeptide that is bound by one or more anti-Gag antibodies;elicits a humoral and/or cellular immune response; and/or exhibits theability to form particles.

An “antigen” refers to a molecule containing one or more epitopes(either linear, conformational or both) that will stimulate a host'simmune system to make a humoral and/or cellular antigen-specificresponse. The term is used interchangeably with the term “immunogen.”Normally, a B-cell epitope will include at least about 5 amino acids butcan be as small as 3-4 amino acids. A T-cell epitope, such as a CTLepitope, will include at least about 7-9 amino acids, and a helperT-cell epitope at least about 12-20 amino acids. Normally, an epitopewill include between about 7 and 15 amino acids, such as, 9, 10, 12 or15 amino acids. The term “antigen” denotes both subunit antigens, (i.e.,antigens which are separate and discrete from a whole organism withwhich the antigen is associated in nature), as well as, killed,attenuated or inactivated bacteria, viruses, fungi, parasites or othermicrobes. Antibodies such as anti-idiotype antibodies, or fragmentsthereof, and synthetic peptide mimotopes, which can mimic an antigen orantigenic determinant, are also captured under the definition of antigenas used herein. Similarly, an oligonucleotide or polynucleotide whichexpresses an antigen or antigenic determinant in vivo, such as in genetherapy and DNA immunization applications, is also included in thedefinition of antigen herein.

For purposes of the present invention, antigens can be derived from anyof several known viruses, bacteria, parasites and fungi, as describedmore fully below. The term also intends any of the various tumorantigens. Furthermore, for purposes of the present invention, an“antigen” refers to a protein which includes modifications, such asdeletions, additions and substitutions (generally conservative innature), to the native sequence, so long as the protein maintains theability to elicit an immunological response, as defined herein. Thesemodifications may be deliberate, as through site-directed mutagenesis,or may be accidental, such as through mutations of hosts which producethe antigens.

An “immunological response” to an antigen or composition is thedevelopment in a subject of a humoral and/or a cellular immune responseto an antigen present in the composition of interest. For purposes ofthe present invention, a “humoral immune response” refers to an immuneresponse mediated by antibody molecules, while a “cellular immuneresponse” is one mediated by T-lymphocytes and/or other white bloodcells. One important aspect of cellular immunity involves anantigen-specific response by cytolytic T-cells (“CTL”s). CTLs havespecificity for peptide antigens that are presented in association withproteins encoded by the major histocompatibility complex (MHC) andexpressed on the surfaces of cells. CTLs help induce and promote thedestruction of intracellular microbes, or the lysis of cells infectedwith such microbes. Another aspect of cellular immunity involves anantigen-specific response by helper T-cells. Helper T-cells act to helpstimulate the function, and focus the activity of, nonspecific effectorcells against cells displaying peptide antigens in association with MHCmolecules on their surface. A “cellular immune response” also refers tothe production of cytokines, chemokines and other such moleculesproduced by activated T-cells and/or other white blood cells, includingthose derived from CD4+ and CD8+ T-cells.

A composition or vaccine that elicits a cellular immune response mayserve to sensitize a vertebrate subject by the presentation of antigenin association with MHC molecules at the cell surface. The cell-mediatedimmune response is directed at, or near, cells presenting antigen attheir surface. In addition, antigen-specific T-lymphocytes can begenerated to allow for the future protection of an immunized host.

The ability of a particular antigen to stimulate a cell-mediatedimmunological response may be determined by a number of assays, such asby lymphoproliferation (lymphocyte activation) assays, CTL cytotoxiccell assays, or by assaying for T-lymphocytes specific for the antigenin a sensitized subject. Such assays are well known in the art. See,e.g., Erickson et al., J. Immunol. (1993) 151:4189-4199; Doe et al.,Eur. J. Immunol. (1994) 24:2369-2376. Recent methods of measuringcell-mediated immune response include measurement of intracellularcytokines or cytokine secretion by T-cell populations, or by measurementof epitope specific T-cells (e.g., by the tetramer technique) (reviewedby McMichael, A. J., and O'Callaghan, C. A., J. Exp. Med.187(9)1367-1371, 1998; Mcheyzer-Williams, M. G., et al, Immunol. Rev.150:5-21, 1996; Lalvani, A., et al, J. Exp. Med. 186:859-865, 1997).

Thus, an immunological response as used herein may be one whichstimulates the production of CTLs, and/or the production or activationof helper T-cells. The antigen of interest may also elicit anantibody-mediated immune response. Hence, an immunological response mayinclude one or more of the following effects: the production ofantibodies by B-cells; and/or the activation of suppressor T-cellsand/or γδ T-cells directed specifically to an antigen or antigenspresent in the composition or vaccine of interest. These responses mayserve to neutralize infectivity, and/or mediate antibody-complement, orantibody dependent cell cytotoxicity (ADCC) to provide protection to animmunized host. Such responses can be determined using standardimmunoassays and neutralization assays, well known in the art.

An “immunogenic composition” is a composition that comprises anantigenic molecule where administration of the composition to a subjectresults in the development in the subject of a humoral and/or a cellularimmune response to the antigenic molecule of interest. The immunogeniccomposition can be introduced directly into a recipient subject, such asby injection, inhalation, oral, intranasal and mucosal (e.g.,intra-rectally or intra-vaginally) administration.

By “subunit vaccine” is meant a vaccine composition which includes oneor more selected antigens but not all antigens, derived from orhomologous to, an antigen from a pathogen of interest such as from avirus, bacterium, parasite or fungus. Such a composition issubstantially free of intact pathogen cells or pathogenic particles, orthe lysate of such cells or particles. Thus, a “subunit vaccine” can beprepared from at least partially purified (preferably substantiallypurified) immunogenic polypeptides from the pathogen, or analogsthereof. The method of obtaining an antigen included in the subunitvaccine can thus include standard purification techniques, recombinantproduction, or synthetic production.

“Substantially purified” general refers to isolation of a substance(compound, polynucleotide, protein, polypeptide, polypeptidecomposition) such that the substance comprises the majority percent ofthe sample in which it resides. Typically in a sample a substantiallypurified component comprises 50%, preferably 80%-85%, more preferably90-95% of the sample. Techniques for purifying polynucleotides andpolypeptides of interest are well-known in the art and include, forexample, ion-exchange chromatography, affinity chromatography andsedimentation according to density.

A “coding sequence” or a sequence which “encodes” a selectedpolypeptide, is a nucleic acid molecule which is transcribed (in thecase of DNA) and translated (in the case of mRNA) into a polypeptide invivo when placed under the control of appropriate regulatory sequences(or “control elements”). The boundaries of the coding sequence aredetermined by a start codon at the 5′ (amino) terminus and a translationstop codon at the 3′ (carboxy) terminus. A coding sequence can include,but is not limited to, cDNA from viral, procaryotic or eucaryotic mRNA,genomic DNA sequences from viral or procaryotic DNA, and even syntheticDNA sequences. A transcription termination sequence such as a stop codonmay be located 3′ to the coding sequence.

Typical “control elements”, include, but are not limited to,transcription promoters, transcription enhancer elements, transcriptiontermination signals, polyadenylation sequences (located 3′ to thetranslation stop codon), sequences for optimization of initiation oftranslation (located 5′ to the coding sequence), and translationtermination sequences. For example, the sequences and/or vectorsdescribed herein may also include one or more additional sequences thatmay optimize translation and/or termination including, but not limitedto, a Kozak sequence (e.g., GCCACC placed in front (5′) of the ATG ofthe codon-optimized wild-type leader or any other suitable leadersequence (e.g., tpa1, tpa2, wtLnat (native wild-type leader)) or atermination sequence (e.g., TAA or, preferably, TAAA placed after (3′)the coding sequence.

A “polynucleotide coding sequence” or a sequence which “encodes” aselected polypeptide, is a nucleic acid molecule which is transcribed(in the case of DNA) and translated (in the case of mRNA) into apolypeptide in vivo when placed under the control of appropriateregulatory sequences (or “control elements”). The boundaries of thecoding sequence are determined by a start codon, for example, at or nearthe 5′ terminus and a translation stop codon, for example, at or nearthe 3′ terminus. Exemplary coding sequences are the modified viralpolypeptide-coding sequences of the present invention. The codingregions of the polynucleotide sequences of the present invention areidentifiable by one of skill in the art and may, for example, be easilyidentified by performing translations of all three frames of thepolynucleotide and identifying the frame corresponding to the encodedpolypeptide, for example, a synthetic nef polynucleotide of the presentinvention encodes a nef-derived polypeptide. A transcription terminationsequence may be located 3′ to the coding sequence. Typical “controlelements”, include, but are not limited to, transcription regulators,such as promoters, transcription enhancer elements, transcriptiontermination signals, and polyadenylation sequences; and translationregulators, such as sequences for optimization of initiation oftranslation, e.g., Shine-Dalgarno (ribosome binding site) sequences,Kozak sequences (i.e., sequences for the optimization of translation,located, for example, 5′ to the coding sequence), leader sequences,translation initiation codon (e.g., ATG), and translation terminationsequences. In certain embodiments, one or more translation regulation orinitiation sequences (e.g., the leader sequence) are derived fromwild-type translation initiation sequences, i.e., sequences thatregulate translation of the coding region in their native state.Wild-type leader sequences that have been modified, using the methodsdescribed herein, also find use in the present invention. Promoters caninclude inducible promoters (where expression of a polynucleotidesequence operably linked to the promoter is induced by an analyte,cofactor, regulatory protein, etc.), repressible promoters (whereexpression of a polynucleotide sequence operably linked to the promoteris induced by an analyte, cofactor, regulatory protein, etc.), andconstitutive promoters.

A “nucleic acid” molecule can include, but is not limited to,procaryotic sequences, eucaryotic mRNA, cDNA from eucaryotic mRNA,genomic DNA sequences from eucaryotic (e.g., mammalian) DNA, and evensynthetic DNA sequences. The term also captures sequences that includeany of the known base analogs of DNA and RNA.

“Operably linked” refers to an arrangement of elements wherein thecomponents so described are configured so as to perform their usualfunction. Thus, a given promoter operably linked to a coding sequence iscapable of effecting the expression of the coding sequence when theproper enzymes are present. The promoter need not be contiguous with thecoding sequence, so long as it functions to direct the expressionthereof. Thus, for example, intervening untranslated yet transcribedsequences can be present between the promoter sequence and the codingsequence and the promoter sequence can still be considered “operablylinked” to the coding sequence.

“Recombinant” as used herein to describe a nucleic acid molecule means apolynucleotide of genomic, cDNA, semisynthetic, or synthetic originwhich, by virtue of its origin or manipulation: (1) is not associatedwith all or a portion of the polynucleotide with which it is associatedin nature; and/or (2) is linked to a polynucleotide other than that towhich it is linked in nature. The term “recombinant” as used withrespect to a protein or polypeptide means a polypeptide produced byexpression of a recombinant polynucleotide. “Recombinant host cells,”“host cells,” “cells,” “cell lines,” “cell cultures,” and other suchterms denoting procaryotic microorganisms or eucaryotic cell linescultured as unicellular entities, are used inter-changeably, and referto cells which can be, or have been, used as recipients for recombinantvectors or other transfer DNA, and include the progeny of the originalcell which has been transfected. It is understood that the progeny of asingle parental cell may not necessarily be completely identical inmorphology or in genomic or total DNA complement to the original parent,due to accidental or deliberate mutation. Progeny of the parental cellwhich are sufficiently similar to the parent to be characterized by therelevant property, such as the presence of a nucleotide sequenceencoding a desired peptide, are included in the progeny intended by thisdefinition, and are covered by the above terms.

Techniques for determining amino acid sequence “similarity” are wellknown in the art. In general, “similarity” means the exact amino acid toamino acid comparison of two or more polypeptides at the appropriateplace, where amino acids are identical or possess similar chemicaland/or physical properties such as charge or hydrophobicity. A so-termed“percent similarity” then can be determined between the comparedpolypeptide sequences. Techniques for determining nucleic acid and aminoacid sequence identity also are well known in the art and includedetermining the nucleotide sequence of the mRNA for that gene (usuallyvia a cDNA intermediate) and determining the amino acid sequence encodedthereby, and comparing this to a second amino acid sequence. In general,“identity” refers to an exact nucleotide to nucleotide or amino acid toamino acid correspondence of two polynucleotides or polypeptidesequences, respectively.

Two or more polynucleotide sequences can be compared by determiningtheir “percent identity.” Two or more amino acid sequences likewise canbe compared by determining their “percent identity.” The percentidentity of two sequences, whether nucleic acid or peptide sequences, isgenerally described as the number of exact matches between two alignedsequences divided by the length of the shorter sequence and multipliedby 100. An approximate alignment for nucleic acid sequences is providedby the local homology algorithm of Smith and Waterman, Advances inApplied Mathematics 2:482-489 (1981). This algorithm can be extended touse with peptide sequences using the scoring matrix developed byDayhoff, Atlas of Protein Sequences and Structure, M. O. Dayhoff ed., 5suppl. 3:353-358, National Biomedical Research Foundation, Washington,D.C., USA, and normalized by Gribskov, Nucl. Acids Res. 14(6):6745-6763(1986). An implementation of this algorithm for nucleic acid and peptidesequences is provided by the Genetics Computer Group (Madison, Wis.) intheir BestFit utility application. The default parameters for thismethod are described in the Wisconsin Sequence Analysis Package ProgramManual, Version 8 (1995) (available from Genetics Computer Group,Madison, Wis.). Other equally suitable programs for calculating thepercent identity or similarity between sequences are generally known inthe art.

For example, percent identity of a particular nucleotide sequence to areference sequence can be determined using the homology algorithm ofSmith and Waterman with a default scoring table and a gap penalty of sixnucleotide positions. Another method of establishing percent identity inthe context of the present invention is to use the MPSRCH package ofprograms copyrighted by the University of Edinburgh, developed by JohnF. Collins and Shane S. Sturrok, and distributed by IntelliGenetics,Inc. (Mountain View, Calif.). From this suite of packages, theSmith-Waterman algorithm can be employed where default parameters areused for the scoring table (for example, gap open penalty of 12, gapextension penalty of one, and a gap of six). From the data generated,the “Match” value reflects “sequence identity.” Other suitable programsfor calculating the percent identity or similarity between sequences aregenerally known in the art, such as the alignment program BLAST, whichcan also be used with default parameters. For example, BLASTN and BLASTPcan be used with the following default parameters: geneticcode=standard; filter=none; strand=both; cutoff=60; expect=10;Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE;Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDStranslations+Swiss protein+Spupdate+PIR. Details of these programs canbe found at the following internet address:http://www.ncbi.nlm.gov/cgi-bin/BLAST.

One of skill in the art can readily determine the proper searchparameters to use for a given sequence, exemplary preferred SmithWaterman based parameters are presented above. For example, the searchparameters may vary based on the size of the sequence in question. Thus,for the polynucleotide sequences of the present invention the length ofthe polynucleotide sequence disclosed herein is searched against aselected database and compared to sequences of essentially the samelength to determine percent identity. For example, a representativeembodiment of the present invention would include an isolatedpolynucleotide comprising X contiguous nucleotides, wherein (i) the Xcontiguous nucleotides have at least about a selected level of percentidentity relative to Y contiguous nucleotides of one or more of thesequences described herein (e.g., in Table C) or fragment thereof, and(ii) for search purposes X equals Y, wherein Y is a selected referencepolynucleotide of defined length (for example, a length of from 15nucleotides up to the number of nucleotides present in a selectedfull-length sequence).

The sequences of the present invention can include fragments of thesequences, for example, from about 15 nucleotides up to the number ofnucleotides present in the full-length sequences described herein (e.g.,see the Figures), including all integer values falling within theabove-described range. For example, fragments of the polynucleotidesequences of the present invention may be 30-60 nucleotides, 60-120nucleotides, 120-240 nucleotides, 240-480 nucleotides, 480-1000nucleotides, and all integer values therebetween.

The synthetic expression cassettes (and purified polynucleotides) of thepresent invention include related polynucleotide sequences having about80% to 100%, greater than 80-85%, preferably greater than 90-92%, morepreferably greater than 95%, and most preferably greater than 98% up to100% (including all integer values falling within these describedranges) sequence identity to the synthetic expression cassette and/orpolynucleotide sequences disclosed herein (for example, to the sequencesof the present invention) when the sequences of the present inventionare used as the query sequence against, for example, a database ofsequences.

Two nucleic acid fragments are considered to “selectively hybridize” asdescribed herein. The degree of sequence identity between two nucleicacid molecules affects the efficiency and strength of hybridizationevents between such molecules. A partially identical nucleic acidsequence will at least partially inhibit a completely identical sequencefrom hybridizing to a target molecule. Inhibition of hybridization ofthe completely identical sequence can be assessed using hybridizationassays that are well known in the art (e.g., Southern blot, Northernblot, solution hybridization, or the like, see Sambrook, et al., supraor Ausubel et al., supra). Such assays can be conducted using varyingdegrees of selectivity, for example, using conditions varying from lowto high stringency. If conditions of low stringency are employed, theabsence of non-specific binding can be assessed using a secondary probethat lacks even a partial degree of sequence identity (for example, aprobe having less than about 30% sequence identity with the targetmolecule), such that, in the absence of non-specific binding events, thesecondary probe will not hybridize to the target.

When utilizing a hybridization-based detection system, a nucleic acidprobe is chosen that is complementary to a target nucleic acid sequence,and then by selection of appropriate conditions the probe and the targetsequence “selectively hybridize,” or bind, to each other to form ahybrid molecule. A nucleic acid molecule that is capable of hybridizingselectively to a target sequence under “moderately stringent” typicallyhybridizes under conditions that allow detection of a target nucleicacid sequence of at least about 10-14 nucleotides in length having atleast approximately 70% sequence identity with the sequence of theselected nucleic acid probe. Stringent hybridization conditionstypically allow detection of target nucleic acid sequences of at leastabout 10-14 nucleotides in length having a sequence identity of greaterthan about 90-95% with the sequence of the selected nucleic acid probe.Hybridization conditions useful for probe/target hybridization where theprobe and target have a specific degree of sequence identity, can bedetermined as is known in the art (see, for example, Nucleic AcidHybridization: A Practical Approach, editors B. D. Hames and S. J.Higgins, (1985) Oxford; Washington, D.C.; IRL Press).

With respect to stringency conditions for hybridization, it is wellknown in the art that numerous equivalent conditions can be employed toestablish a particular stringency by varying, for example, the followingfactors: the length and nature of probe and target sequences, basecomposition of the various sequences, concentrations of salts and otherhybridization solution components, the presence or absence of blockingagents in the hybridization solutions (e.g., formamide, dextran sulfate,and polyethylene glycol), hybridization reaction temperature and timeparameters, as well as, varying wash conditions. The selection of aparticular set of hybridization conditions is selected followingstandard methods in the art (see, for example, Sambrook, et al., supraor Ausubel et al., supra).

A first polynucleotide is “derived from” second polynucleotide if it hasthe same or substantially the same basepair sequence as a region of thesecond polynucleotide, its cDNA, complements thereof, or if it displayssequence identity as described above.

A first polypeptide is “derived from” a second polypeptide if it is (i)encoded by a first polynucleotide derived from a second polynucleotide,or (ii) displays sequence identity to the second polypeptides asdescribed above.

Generally, a viral polypeptide is “derived from” a particularpolypeptide of a virus (viral polypeptide) if it is (i) encoded by anopen reading frame of a polynucleotide of that virus (viralpolynucleotide), or (ii) displays sequence identity to polypeptides ofthat virus as described above.

“Encoded by” refers to a nucleic acid sequence which codes for apolypeptide sequence, wherein the polypeptide sequence or a portionthereof contains an amino acid sequence of at least 3 to 5 amino acids,more preferably at least 8 to 10 amino acids, and even more preferablyat least 15 to 20 amino acids from a polypeptide encoded by the nucleicacid sequence. Also encompassed are polypeptide sequences which areimmunologically identifiable with a polypeptide encoded by the sequence.Further, polyproteins can be constructed by fusing in-frame two or morepolynucleotide sequences encoding polypeptide or peptide products.Further, polycistronic coding sequences may be produced by placing twoor more polynucleotide sequences encoding polypeptide products adjacenteach other, typically under the control of one promoter, wherein eachpolypeptide coding sequence may be modified to include sequences forinternal ribosome binding sites.

“Purified polynucleotide” refers to a polynucleotide of interest orfragment thereof which is essentially free, e.g., contains less thanabout 50%, preferably less than about 70%, and more preferably less thanabout 90%, of the protein with which the polynucleotide is naturallyassociated. Techniques for purifying polynucleotides of interest arewell-known in the art and include, for example, disruption of the cellcontaining the polynucleotide with a chaotropic agent and separation ofthe polynucleotide(s) and proteins by ion-exchange chromatography,affinity chromatography and sedimentation according to density.

By “nucleic acid immunization” is meant the introduction of a nucleicacid molecule encoding one or more selected antigens into a host cell,for the in vivo expression of an antigen, antigens, an epitope, orepitopes. The nucleic acid molecule can be introduced directly into arecipient subject, such as by injection, inhalation, oral, intranasaland mucosal administration, or the like, or can be introduced ex vivo,into cells which have been removed from the host. In the latter case,the transformed cells are reintroduced into the subject where an immuneresponse can be mounted against the antigen encoded by the nucleic acidmolecule.

“Gene transfer” or “gene delivery” refers to methods or systems forreliably inserting DNA of interest into a host cell. Such methods canresult in transient expression of non-integrated transferred DNA,extrachromosomal replication and expression of transferred replicons(e.g., episomes), or integration of transferred genetic material intothe genomic DNA of host cells. Gene delivery expression vectors include,but are not limited to, vectors derived from alphaviruses, pox virusesand vaccinia viruses. When used for immunization, such gene deliveryexpression vectors may be referred to as vaccines or vaccine vectors.

“T lymphocytes” or “T cells” are non-antibody producing lymphocytes thatconstitute a part of the cell-mediated arm of the immune system. T cellsarise from immature lymphocytes that migrate from the bone marrow to thethymus, where they undergo a maturation process under the direction ofthymic hormones. Here, the mature lymphocytes rapidly divide increasingto very large numbers. The maturing T cells become immunocompetent basedon their ability to recognize and bind a specific antigen. Activation ofimmunocompetent T cells is triggered when an antigen binds to thelymphocyte's surface receptors.

The term “transfection” is used to refer to the uptake of foreign DNA bya cell. A cell has been “transfected” when exogenous DNA has beenintroduced inside the cell membrane. A number of transfection techniquesare generally known in the art. See, e.g., Graham et al. (1973)Virology, 52:456, Sambrook et al. (1989) Molecular Cloning, a laboratorymanual, Cold Spring Harbor Laboratories, New York, Davis et al. (1986)Basic Methods in Molecular Biology, Elsevier, and Chu et al. (1981) Gene13:197. Such techniques can be used to introduce one or more exogenousDNA moieties into suitable host cells. The term refers to both stableand transient uptake of the genetic material, and includes uptake ofpeptide- or antibody-linked DNAs.

A “vector” is capable of transferring gene sequences to target cells(e.g., viral vectors, non-viral vectors, particulate carriers, andliposomes). Typically, “vector construct,” “expression vector,” and“gene transfer vector,” mean any nucleic acid construct capable ofdirecting the expression of a gene of interest and which can transfergene sequences to target cells. Thus, the term includes cloning andexpression vehicles, as well as viral vectors.

Transfer of a “suicide gene” (e.g., a drug-susceptibility gene) to atarget cell renders the cell sensitive to compounds or compositions thatare relatively nontoxic to normal cells. Moolten, F. L. (1994) CancerGene Ther. 1:279-287. Examples of suicide genes are thymidine kinase ofherpes simplex virus (HSV-tk), cytochrome P450 (Manome et al. (1996)Gene Therapy 3:513-520), human deoxycytidine kinase (Manome et al.(1996) Nature Medicine 2(5):567-573) and the bacterial enzyme cytosinedeaminase (Dong et al. (1996) Human Gene Therapy 7:713-720). Cells whichexpress these genes are rendered sensitive to the effects of therelatively nontoxic prodrugs ganciclovir (HSV-tk), cyclophosphamide(cytochrome P450 2B1), cytosine arabinoside (human deoxycytidine kinase)or 5-fluorocytosine (bacterial cytosine deaminase). Culver et al. (1992)Science 256:1550-1552, Huber et al. (1994) Proc. Natl. Acad. Sci. USA91:8302-8306.

A “selectable marker” or “reporter marker” refers to a nucleotidesequence included in a gene transfer vector that has no therapeuticactivity, but rather is included to allow for simpler preparation,manufacturing, characterization or testing of the gene transfer vector.

A “specific binding agent” refers to a member of a specific binding pairof molecules wherein one of the molecules specifically binds to thesecond molecule through chemical and/or physical means. One example of aspecific binding agent is an antibody directed against a selectedantigen.

By “subject” is meant any member of the subphylum chordata, including,without limitation, humans and other primates, including non-humanprimates such as rhesus macaque, chimpanzees and other apes and monkeyspecies; farm animals such as cattle, sheep, pigs, goats and horses;domestic mammals such as dogs and cats; laboratory animals includingrodents such as mice, rats and guinea pigs; birds, including domestic,wild and game birds such as chickens, turkeys and other gallinaceousbirds, ducks, geese, and the like. The term does not denote a particularage. Thus, both adult and newborn individuals are intended to becovered. The system described above is intended for use in any of theabove vertebrate species, since the immune systems of all of thesevertebrates operate similarly.

By “pharmaceutically acceptable” or “pharmacologically acceptable” ismeant a material which is not biologically or otherwise undesirable,i.e., the material may be administered to an individual in a formulationor composition without causing any undesirable biological effects orinteracting in a deleterious manner with any of the components of thecomposition in which it is contained.

By “physiological pH” or a “pH in the physiological range” is meant a pHin the range of approximately 7.0 to 8.0 inclusive, more typically inthe range of approximately 7.2 to 7.6 inclusive.

As used herein, “treatment” refers to any of (i) the prevention ofinfection or reinfection, as in a traditional vaccine, (ii) thereduction or elimination of symptoms, and (iii) the substantial orcomplete elimination of the pathogen in question. Treatment may beeffected prophylactically (prior to infection) or therapeutically(following infection).

By “co-administration” is meant administration of more than onecomposition or molecule. Thus, co-administration includes concurrentadministration or sequentially administration (in any order), via thesame or different routes of administration. Non-limiting examples ofco-administration regimes include, co-administration of nucleic acid andpolypeptide; co-administration of different nucleic acids (e.g.,different expression cassettes as described herein and/or different genedelivery vectors); and co-administration of different polypeptides(e.g., different HIV polypeptides and/or different adjuvants). The termalso encompasses multiple administrations of one of the co-administeredmolecules or compositions (e.g., multiple administrations of one or moreof the expression cassettes described herein followed by one or moreadministrations of a polypeptide-containing composition). In cases wherethe molecules or compositions are delivered sequentially, the timebetween each administration can be readily determined by one of skill inthe art in view of the teachings herein.

“Lentiviral vector”, and “recombinant lentiviral vector” refer to anucleic acid construct which carries, and within certain embodiments, iscapable of directing the expression of a nucleic acid molecule ofinterest. The lentiviral vector include at least one transcriptionalpromoter/enhancer or locus defining element(s), or other elements whichcontrol gene expression by other means such as alternate splicing,nuclear RNA export, post-translational modification of messenger, orpost-transcriptional modification of protein. Such vector constructsmust also include a packaging signal, long terminal repeats (LTRS) orportion thereof, and positive and negative strand primer binding sitesappropriate to the retrovirus used (if these are not already present inthe retroviral vector). Optionally, the recombinant lentiviral vectormay also include a signal which directs polyadenylation, selectablemarkers such as Neo, TK, hygromycin, phleomycin, histidinol, or DHFR, aswell as one or more restriction sites and a translation terminationsequence. By way of example, such vectors typically include a 5′ LTR, atRNA binding site, a packaging signal, an origin of second strand DNAsynthesis, and a 3′LTR or a portion thereof “Lentiviral vector particle”as utilized within the present invention refers to a lentivirus whichcarries at least one gene of interest. The retrovirus may also contain aselectable marker. The recombinant lentivirus is capable of reversetranscribing its genetic material (RNA) into DNA and incorporating thisgenetic material into a host cell's DNA upon infection. Lentiviralvector particles may have a lentiviral envelope, a non-lentiviralenvelope (e.g., an ampho or VSV-G envelope), or a chimeric envelope.

“Nucleic acid expression vector” or “Expression cassette” refers to anassembly which is capable of directing the expression of a sequence orgene of interest. The nucleic acid expression vector includes a promoterwhich is operably linked to the sequences or gene(s) of interest. Othercontrol elements may be present as well. Expression cassettes describedherein may be contained within a plasmid construct. In addition to thecomponents of the expression cassette, the plasmid construct may alsoinclude a bacterial origin of replication, one or more selectablemarkers, a signal which allows the plasmid construct to exist assingle-stranded DNA (e.g., a M13 origin of replication), a multiplecloning site, and a “mammalian” origin of replication (e.g., a SV40 oradenovirus origin of replication).

“Packaging cell” refers to a cell which contains those elementsnecessary for production of infectious recombinant retrovirus which arelacking in a recombinant retroviral vector. Typically, such packagingcells contain one or more expression cassettes which are capable ofexpressing proteins which encode Gag, pol and env proteins.

“Producer cell” or “vector producing cell” refers to a cell whichcontains all elements necessary for production of recombinant retroviralvector particles.

2. MODES OF CARRYING OUT THE INVENTION

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to particular formulationsor process parameters as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments of the invention only, and is notintended to be limiting.

Although a number of methods and materials similar or equivalent tothose described herein can be used in the practice of the presentinvention, the preferred materials and methods are described herein.

2.1. The HIV Genome

The HIV genome and various polypeptide-encoding regions are shown inTable A. The nucleotide positions are given relative to 8_(—)5_TV1—C.ZA(FIG. 1; an HIV Type C isolate). However, it will be readily apparent toone of ordinary skill in the art in view of the teachings of the presentdisclosure how to determine corresponding regions in other HIV strainsor variants (e.g., isolates HIV_(IIIb), HIV_(SF2), HIV-1_(SF162),HIV-1_(SF170), HIV_(LAV), HIV_(MN), HIV_(CM235), HIV-1_(US4), otherHIV-1 strains from diverse subtypes (e.g., subtypes, A through G, andO), HIV-2 strains and diverse subtypes (e.g., HIV-2_(UC1), andHIV-2_(UC2)), and simian immunodeficiency virus (SIV). (See, e.g.,Virology, 3rd Edition (W. K. Joklik ed. 1988); Fundamental Virology, 2ndEdition (B. N. Fields and D. M. Knipe, eds. 1991); Virology, 3rd Edition(Fields, B N, D M Knipe, P M Howley, Editors, 1996, Lippincott-Raven,Philadelphia, Pa.; for a description of these and other relatedviruses), using for example, sequence comparison programs (e.g., BLASTand others described herein) or identification and alignment ofstructural features (e.g., a program such as the “ALB” program describedherein that can identify the various regions). TABLE A Regions of theHIV Genome relative to 8_5_TV1_C.ZA Region Position in nucleotidesequence 5′LTR  1-636 U3  1-457 R 458-553 U5 554-636 NFkB II 340-348NFkB I 354-362 Sp1 III 379-388 Sp1 II 390-398 Sp1 I 400-410 TATA Box429-433 TAR 474-499 Poly A signal 529-534 PBS 638-655 p7 binding region,packaging signal 685-791 Gag:  792-2285 p17  792-1178 p24 1179-1871Cyclophilin A bdg. 1395-1505 MHR 1632-1694 p2 1872-1907 p7 1908-2072Frameshift slip 2072-2078 p1 2073-2120 p6Gag 2121-2285 Zn-motif I1950-1991 Zn-motif II 2013-2054 Pol: 2072-5086 p6Pol 2072-2245 Prot2246-2542 p66RT 2543-4210 p15RNaseH 3857-4210 p31Int 4211-5086 Vif:5034-5612 Hydrophilic region 5292-5315 Vpr: 5552-5839 Oligomerization5552-5677 Amphipathic a-helix 5597-5653 Tat: 5823-6038 and 8417-8509Tat-1 exon 5823-6038 Tat-2 exon 8417-8509 N-terminal domain 5823-5885Trans-activation domain 5886-5933 Transduction domain 5961-5993 Rev:5962-6037 and 8416-8663 Rev-1 exon 5962-6037 Rev-2 exon 8416-8663High-affinity bdg. site 8439-8486 Leu-rich effector domain 8562-8588Vpu: 6060-6326 Transmembrane domain 6060-6161 Cytoplasmic domain6162-6326 Env (gp160): 6244-8853 Signal peptide 6244-6324 gp1206325-7794 V1 6628-6729 V2 6727-6852 V3 7150-7254 V4 7411-7506 V57663-7674 C1 6325-6627 C2 6853-7149 C3 7255-7410 C4 7507-7662 C57675-7794 CD4 binding 7540-7566 gp41 7795-8853 Fusion peptide 7789-7842Oligomerization domain 7924-7959 N-terminal heptad repeat 7921-8028C-terminal heptad repeat 8173-8280 Immunodominant region 8023-8076 Nef:8855-9478 Myristoylation 8858-8875 SH3 binding 9062-9091 Polypurinetract 9128-9154 SH3 binding 9296-9307

It will be readily apparent that one of skill in the art can readilyalign any sequence to that shown in Table A to determine relativelocations of any particular HIV gene. For example, using one of thealignment programs described herein (e.g., BLAST), other HIV genomicsequences can be aligned with 8_(—)5_TV1—C.ZA (Table A) and locations ofgenes determined. Polypeptide sequences can be similarly aligned. Forexample, FIGS. 2A-2C shows the alignment of Env polypeptide sequencesfrom various strains, relative to SF-162. As described in detail inco-owned WO/39303 (herein incorporated by reference), Env polypeptides(e.g., gp120, gp140 and gp160) include a “bridging sheet” comprised of 4anti-parallel b-strands (b-2, b-3, b-20 and b-21) that form a b-sheet.Extruding from one pair of the b-strands (b-2 and b-3) are two loops, V1and V2. The b-2 sheet occurs at approximately amino acid residue 113(Cys) to amino acid residue 117 (Thr) while b-3 occurs at approximatelyamino acid residue 192 (Ser) to amino acid residue 194 (Ile), relativeto SF-162. The “V1/V2 region” occurs at approximately amino acidpositions 120 (Cys) to residue 189 (Cys), relative to SF-162. Extrudingfrom the second pair of b-strands (b-20 and b-21) is a “small-loop”structure, also referred to herein as “the bridging sheet small loop.”The locations of both the small loop and bridging sheet small loop canbe determined relative to HXB-2 following the teachings herein and inWO/39303. Also shown by arrows in FIG. 2A-C are approximate sites fordeletions sequence from the beta sheet region. The “*” denotesN-glycosylation sites that can be mutated following the teachings of thepresent specification.

2.2.0 Synthetic Expression Cassettes

One aspect of the present invention is the generation of HIV-1 codingsequences, and related sequences, for example having improved expressionrelative to the corresponding wild-type sequences.

2.2.1 Modification of HIV-1 Nucleic Acid Coding Sequences

First, the HIV-1 codon usage pattern was modified so that the resultingnucleic acid coding sequence was comparable to codon usage found inhighly expressed human genes. The HIV codon usage reflects a highcontent of the nucleotides A or T of the codon-triplet. The effect ofthe HIV-1 codon usage is a high AT content in the DNA sequence thatresults in a decreased translation ability and instability of the mRNA.In comparison, highly expressed human codons prefer the nucleotides G orC. The HIV coding sequences were modified to be comparable to codonusage found in highly expressed human genes.

Second, there are inhibitory (or instability) elements (INS) locatedwithin the coding sequences of, for example, the Gag coding sequences.The RRE is a secondary RNA structure that interacts with the HIV encodedRev-protein to overcome the expression down-regulating effects of theINS. To overcome the post-transcriptional activating mechanisms of RREand Rev, the instability elements can be inactivated by introducingmultiple point mutations that do not alter the reading frame of theencoded proteins.

Third, for some genes the coding sequence has been altered such that thepolynucleotide coding sequence encodes a gene product that is inactiveor non-functional (e.g., inactivated polymerase, protease, tat, rev,nef, vif, vpr, and/or vpu gene products). Example 1 describes someexemplary mutations. Example 8 presents information concerningfunctional analysis of mutated Tat, Rev and Nef antigens.

The synthetic coding sequences are assembled by methods known in theart, for example by companies such as the Midland Certified ReagentCompany (Midland, Tex.).

Modification of the Gag polypeptide coding sequences results in improvedexpression relative to the wild-type coding sequences in a number ofmammalian cell lines (as well as other types of cell lines, including,but not limited to, insect cells).

Some exemplary polynucleotide sequences encoding Gag-containingpolypeptides are GagComplPolmut.SF2, GagComplPolmutAtt.SF2,GagComplPolmutIna.SF2, gagCpolInaTatRevNef.opt_B, GagPolmutAtt.SF2,GagPolmutIna.SF2, GagProtInaRTmut.SF2, GagProtInaRTmutTatRevNef.opt_B,GagRTmut.SF2, and GagTatRevNef.opt_B.

Similarly, the present invention also includes synthetic Env-encodingpolynucleotides and modified Env proteins, for example, gp140.modSF162.CwtLmod, gp 140.modSF162.CwtLnat, gp160.modSF162.delV2.mut7, and gp 160.modSF162.delV2.mut8.

The codon usage pattern for Env was modified as described above for Gagso that the resulting nucleic acid coding sequence was comparable tocodon usage found in highly expressed human genes. Experiments performedin support of the present invention show that the synthetic Envsequences were capable of higher level of protein production relative tothe native Env sequences.

Modification of the Env polypeptide coding sequences results in improvedexpression relative to the wild-type coding sequences in a number ofmammalian cell lines (as well as other types of cell lines, including,but not limited to, insect cells). Similar Env polypeptide codingsequences can be obtained, modified and tested for improved expressionfrom a variety of isolates, including those described above for Gag.

Further modifications of Env include, but are not limited to, generatingpolynucleotides that encode Env polypeptides having mutations and/ordeletions therein. For instance, the hypervariable regions, V1 and/orV2, can be deleted as described herein. Additionally, othermodifications, for example to the bridging sheet region and/or toN-glycosylation sites within Env can also be performed following theteachings of the present specification. (see, FIG. 2A-C, as well as WO00/39303, WO 00/39302, WO 00/39304, WO 02/04493 all herein incorporatedby reference in their entireties). Various combinations of thesemodifications can be employed to generate synthetic expression cassettesas described herein.

The present invention also includes expression cassettes which includesynthetic Pol sequences. As noted above, “Pol” includes, but is notlimited to, the protein-encoding regions comprising polymerase,protease, reverse transcriptase and/or integrase-containing sequences(Wan et al (1996) Biochem. J. 316:569-573; Kohl et al. (1988) PNAS USA85:4686-4690; Krausslich et al. (1988) J. Virol. 62:4393-4397; Coffin,“Retroviridae and their Replication” in Virology, pp 1437-1500 (Raven,N.Y., 1990); Patel et. al. (1995) Biochemistry 34:5351-5363). Thus, thesynthetic expression cassettes exemplified herein include one or more ofthese regions and one or more changes to the resulting amino acidsequences. Some exemplary polynucleotide sequences encoding Pol-derivedpolypeptides are presented in Table C.

The codon usage pattern for Pol was modified as described above for Gagand Env so that the resulting nucleic acid coding sequence wascomparable to codon usage found in highly expressed human genes.

Constructs may be modified in various ways. For example, the expressionconstructs may include a sequence that encodes the first 6 amino acidsof the integrase polypeptide. This 6 amino acid region is believed toprovide a cleavage recognition site recognized by HIV protease (see,e.g., McCornack et al. (1997) FEBS Letts 414:84-88). Constructs mayinclude a multiple cloning site (MCS) for insertion of one or moretransgenes, typically at the 3′ end of the construct. In addition, acassette encoding a catalytic center epitope derived from the catalyticcenter in RT is typically included 3′ of the sequence encoding 6 aminoacids of integrase. This cassette encodes Ile178 through Serine 191 ofRT and may be added to keep this well conserved region as a possible CTLepitope. Further, the constructs contain an insertion mutations topreserve the reading frame. (see, e.g., Park et al. (1991) J. Virol.65:5111).

In certain embodiments, the catalytic center and/or primer grip regionof RT are modified. The catalytic center and primer grip regions of RTare described, for example, in Patel et al. (1995) Biochem. 34:5351 andPalaniappan et al. (1997) J. Biol. Chem. 272(17): 11157. For example,wild type sequence encoding the amino acids YMDD at positions 183-185 ofp66 RT, numbered relative to AF110975, may be replaced with sequenceencoding the amino acids “AP”. Further, the primer grip region (aminoacids WMGY, residues 229-232 of p66RT, numbered relative to AF110975)may be replaced with sequence encoding the amino acids “PI.”

For the Pol sequence, the changes in codon usage are typicallyrestricted to the regions up to the −1 frameshift and starting again atthe end of the Gag reading frame; however, regions within the frameshifttranslation region can be modified as well. Finally, inhibitory (orinstability) elements (INS) located within the coding sequences of theprotease polypeptide coding sequence can be altered as well.

Experiments can be performed in support of the present invention to showthat the synthetic Pol sequences were capable of higher level of proteinproduction relative to the native Pol sequences. Modification of the Polpolypeptide coding sequences results in improved expression relative tothe wild-type coding sequences in a number of mammalian cell lines (aswell as other types of cell lines, including, but not limited to, insectcells). Similar Pol polypeptide coding sequences can be obtained,modified and tested for improved expression from a variety of isolates,including those described above for Gag and Env.

The present invention also includes expression cassettes which includesynthetic sequences derived HIV genes other than Gag, Env and Pol,including but not limited to, regions within Gag, Env, Pol, as well as,GagComplPolmut.SF2, GagComplPolmutAtt.SF2, GagComplPolmutIna.SF2,gagCpolInaTatRevNef.opt_B, GagPolmutAtt.SF2, GagPolmutIna.SF2,GagProtInaRTmut.SF2, GagProtInaRTmutTatRevNef.opt_B, GagRTmut.SF2,GagTatRevNef.opt_B, gp 140.modSF162.CwtLmod, gp 140.modSF162.CwtLnat, gp160.modSF162.delV2.mut7, gp 160.modSF162.delV2.mut8, int.opt.mut.SF2,int.opt.SF2, nef.D 125G.-myr.opt.SF162, nef.D107G.-myr18.opt.SF162,nef.opt.D125G.SF162, nef.opt.SF162, p15RnaseH.opt.SF2,p2Pol.opt.YMWM.SF2, p2PolInaopt.YM.SF2, p2Polopt.SF2,p2PolTatRevNef.opt.native_B, p2PolTatRevNef.opt_B, pol.opt.SF2,prot.opt.SF2, protIna.opt.SF2, protInaRT.YM.opt.SF2,protInaRT.YMWM.opt.SF2, ProtInaRTmut.SF2, protRT.opt.SF2,ProtRT.TatRevNef.opt_B, ProtRTTatRevNef.opt_B, rev.exon1_(—)2.M5-10.opt.SF162, rev.exon1_(—)2.opt.SF162, RT.opt.SF2 (mutant),RT.opt.SF2 (native), RTmut.SF2, tat.exon1_(—)2.opt.C22-37.5F2,tat.exon1_(—)2.opt.C37.5F2, TatRevNef.opt.native.SF162,TatRevNef.opt.SF162, TatRevNefGag B, TatRevNefgagCpolIna B,TatRevNefGagProtInaRTmut B, TatRevNefp2Pol.opt_B, TatRevNefprotRTopt B,vif.opt.SF2, vpr.opt.SF2, and vpu.opt.SF162. Sequences obtained fromother strains can be manipulated in similar fashion following theteachings of the present specification. As noted above, the codon usagepattern is modified as described above for Gag, Env and Pol so that theresulting nucleic acid coding sequence is comparable to codon usagefound in highly expressed human genes. Typically these syntheticsequences are capable of higher level of protein production relative tothe native sequences and that modification of the wild-type polypeptidecoding sequences results in improved expression relative to thewild-type coding sequences in a number of mammalian cell lines (as wellas other types of cell lines, including, but not limited to, insectcells). Furthermore, the nucleic acid sequence can also be modified tointroduce mutations into one or more regions of the gene, for instanceto alter the function of the gene product (e.g., render the gene productnon-functional) and/or to eliminate site modifications (e.g., themyristoylation site in Nef).

Synthetic expression cassettes, derived from HIV Type B codingsequences, exemplified herein include, but are not limited to, thosecomprising one or more of the following synthetic polynucleotides:GagComplPolmut.SF2, GagComplPolmutAtt.SF2, GagComplPolmutIna.SF2,gagCpolInaTatRevNef.opt_B, GagPolmutAtt.SF2, GagPolmutIna.SF2,GagProtInaRTmut.SF2, GagProtInaRTmutTatRevNef.opt_B, GagRTmut.SF2,GagTatRevNef.opt_B, gp140.modSF162.CwtLmod, gp 140.modSF162.CwtLnat, gp160.modSF162.delV2.mut7, gp 160.modSF162.delV2.mut8, int.opt.mut.SF2,int.opt.SF2, nef.D125G.-myr.opt.SF162, nef.D107G.-myr18.opt.SF162,nef.opt.D125G.SF162, nef.opt.SF162, p15RnaseH.opt.SF2,p2Pol.opt.YMWM.SF2, p2PolInaopt.YM.SF2, p2Polopt.SF2,p2PolTatRevNef.opt.native_B, p2PolTatRevNef.opt_B, pol.opt.SF2,prot.opt.SF2, protIna.opt.SF2, protInaRT.YM.opt.SF2,protInaRT.YMWM.opt.SF2, ProtInaRTmut.SF2, protRT.opt.SF2,ProtRT.TatRevNef.opt_B, ProtRTTatRevNef.opt_B,rev.exon1_(—)2.M5-10.opt.SF162, rev.exon 1_(—)2.opt.SF162, RT.opt.SF2(mutant), RT.opt.SF2 (native), RTmut.SF2, tat.exon1_(—)2.opt.C22-37.5F2,tat.exon 1_(—)2.opt.C37.SF2, TatRevNef.opt.native.SF162,TatRevNef.opt.SF162, TatRevNefGag B, TatRevNefgagCpollna B,TatRevNefGagProtInaRTmut B, TatRevNefp2Pol.opt_B, TatRevNefprotRTopt B,vif.opt.SF2, vpr.opt.SF2, and vpu.opt.SF162.

Gag-complete refers to in-frame polyproteins comprising, e.g., Gag andpol, wherein the p6 portion of Gag is present.

Additional sequences that may be employed in some aspects of the presentinvention have been described in WO 00/39302, WO 00/39303, WO 00/39304,and WO 02/04493, all of which are herein incorporated by reference intheir entireties.

2.2.2 Further Modification of Sequences Including HIV Nucleic AcidCoding Sequences

The HIV polypeptide-encoding expression cassettes described herein mayalso contain one or more further sequences encoding, for example, one ormore transgenes. Further sequences (e.g., transgenes) useful in thepractice of the present invention include, but are not limited to,further sequences are those encoding further viral epitopes/antigens{including but not limited to, HCV antigens (e.g., E1, E2; Houghton, M.,et al., U.S. Pat. No. 5,714,596, issued Feb. 3, 1998; Houghton, M., etal., U.S. Pat. No. 5,712,088, issued Jan. 27, 1998; Houghton, M., etal., U.S. Pat. No. 5,683,864, issued Nov. 4, 1997; Weiner, A. J., etal., U.S. Pat. No. 5,728,520, issued Mar. 17, 1998; Weiner, A. J., etal., U.S. Pat. No. 5,766,845, issued Jun. 16, 1998; Weiner, A. J., etal., U.S. Pat. No. 5,670,152, issued Sep. 23, 1997; all hereinincorporated by reference), HIV antigens (e.g., derived from one or moreHIV isolate); and sequences encoding tumor antigens/epitopes. Furthersequences may also be derived from non-viral sources, for instance,sequences encoding cytokines such interleukin-2 (IL-2), stem cell factor(SCF), interleukin 3 (IL-3), interleukin 6 (IL-6), interleukin 12(IL-12), G-CSF, granulocyte macrophage-colony stimulating factor(GM-CSF), interleukin-1 alpha (IL-1I), interleukin-11 (IL-11), MIP-1I,tumor necrosis factor (TNF), leukemia inhibitory factor (LIF), c-kitligand, thrombopoietin (TPO) and flt3 ligand, commercially availablefrom several vendors such as, for example, Genzyme (Framingham, Mass.),Genentech (South San Francisco, Calif.), Amgen (Thousand Oaks, Calif.),R&D Systems and Immunex (Seattle, Wash.). Additional sequences aredescribed below. Also, variations on the orientation of the Gag andother coding sequences, relative to each other, are described below.

HIV polypeptide coding sequences can be obtained from other HIVisolates, see, e.g., Myers et al. Los Alamos Database, Los AlamosNational Laboratory, Los Alamos, N. Mex. (1992); Myers et al., HumanRetroviruses and Aids, 1997, Los Alamos, N. Mex.: Los Alamos NationalLaboratory. Synthetic expression cassettes can be generated using suchcoding sequences as starting material by following the teachings of thepresent specification.

Further, the synthetic expression cassettes of the present inventioninclude related polypeptide sequences having greater than 85%,preferably greater than 90%, more preferably greater than 95%, and mostpreferably greater than 98% sequence identity to the polypeptidesencoded by the synthetic expression cassette sequences disclosed herein.

Exemplary expression cassettes and modifications are set forth inExample 1.

2.2.3 Expression of Synthetic Sequences Encoding HIV-1 Polypeptides andRelated Polypeptides

Synthetic HIV-encoding sequences (expression cassettes) of the presentinvention can be cloned into a number of different expression vectors toevaluate levels of expression and, in the case of Gag-containingconstructs, production of VLPs. The synthetic DNA fragments for HIVpolypeptides can be cloned into eucaryotic expression vectors,including, a transient expression vector, CMV-promoter-based mammalianvectors, and a shuttle vector for use in baculovirus expression systems.Corresponding wild-type sequences can also be cloned into the samevectors.

These vectors can then be transfected into a several different celltypes, including a variety of mammalian cell lines (293, RD, COS-7, andCHO, cell lines available, for example, from the A.T.C.C.). The celllines are then cultured under appropriate conditions and the levels ofany appropriate polypeptide product can be evaluated in supernatants.(see, Table A). For example, p24 can be used to evaluate Gag expression;gp160, gp140 or gp120 can be used to evaluate Env expression; p6pol canbe used to evaluate Pol expression; prot can be used to evaluateprotease; p15 for RNAseH; p31 for Integrase; and other appropriatepolypeptides for Vif, Vpr, Tat, Rev, Vpu and Nef. Further, modifiedpolypeptides can also be used, for example, other Env polypeptidesinclude, but are not limited to, for example, native gp160, oligomericgp140, monomeric gp120 as well as modified and/or synthetic sequences ofthese polypeptides. The results of these assays demonstrate thatexpression of synthetic HIV polypeptide-encoding sequences aresignificantly higher than corresponding wild-type sequences.

Further, Western Blot analysis can be used to show that cells containingthe synthetic expression cassette produce the expected protein at higherper-cell concentrations than cells containing the native expressioncassette. The HIV proteins can be seen in both cell lysates andsupernatants. The levels of production are significantly higher in cellsupernatants for cells transfected with the synthetic expressioncassettes of the present invention.

Fractionation of the supernatants from mammalian cells transfected withthe synthetic expression cassette can be used to show that the cassettesprovide superior production of HIV proteins and, in the case of Gag,VLPs, relative to the wild-type sequences.

Efficient expression of these HIV-containing polypeptides in mammaliancell lines provides the following benefits: the polypeptides are free ofbaculovirus contaminants; production by established methods approved bythe FDA; increased purity; greater yields (relative to native codingsequences); and a novel method of producing the Sub HIV-containingpolypeptides in CHO cells which is not feasible in the absence of theincreased expression obtained using the constructs of the presentinvention. Exemplary Mammalian cell lines include, but are not limitedto, BHK, VERO, HT1080, 293, 293T, RD, COS-7, CHO, Jurkat, HUT, SUPT,C8166, MOLT4/clone8, MT-2, MT-4, H9, PM1, CEM, and CEMX174 (such celllines are available, for example, from the A.T.C.C.).

A synthetic Gag expression cassette of the present invention will alsoexhibit high levels of expression and VLP production when transfectedinto insect cells. Synthetic expression cassettes described herein alsodemonstrate high levels of expression in insect cells. Further, inaddition to a higher total protein yield, the final product from thesynthetic polypeptides consistently contains lower amounts ofcontaminating baculovirus proteins than the final product from thenative sequences.

Further, synthetic expression cassettes of the present invention canalso be introduced into yeast vectors which, in turn, can be transformedinto and efficiently expressed by yeast cells (Saccharomyces cerevisea;using vectors as described in Rosenberg, S. and Tekamp-Olson, P., U.S.Pat. No. RE35,749, issued, Mar. 17, 1998, herein incorporated byreference).

In addition to the mammalian and insect vectors, the syntheticexpression cassettes of the present invention can be incorporated into avariety of expression vectors using selected expression controlelements. Appropriate vectors and control elements for any given cell anbe selected by one having ordinary skill in the art in view of theteachings of the present specification and information known in the artabout expression vectors.

For example, a synthetic expression cassette can be inserted into avector which includes control elements operably linked to the desiredcoding sequence, which allow for the expression of the gene in aselected cell-type. For example, typical promoters for mammalian cellexpression include the SV40 early promoter, a CMV promoter such as theCMV immediate early promoter (a CMV promoter can include intron A), RSV,HIV-Ltr, the mouse mammary tumor virus LTR promoter (MMLV-ltr), theadenovirus major late promoter (Ad MLP), and the herpes simplex viruspromoter, among others. Other nonviral promoters, such as a promoterderived from the murine metallothionein gene, will also find use formammalian expression. Typically, transcription termination andpolyadenylation sequences will also be present, located 3′ to thetranslation stop codon. Preferably, a sequence for optimization ofinitiation of translation, located 5′ to the coding sequence, is alsopresent. Examples of transcription terminator/polyadenylation signalsinclude those derived from SV40, as described in Sambrook, et al.,supra, as well as a bovine growth hormone terminator sequence. Introns,containing splice donor and acceptor sites, may also be designed intothe constructs for use with the present invention (Chapman et al., Nuc.Acids Res. (1991) 19:3979-3986).

Enhancer elements may also be used herein to increase expression levelsof the mammalian constructs. Examples include the SV40 early geneenhancer, as described in Dijkema et al., EMBO J. (1985) 4:761, theenhancer/promoter derived from the long terminal repeat (LTR) of theRous Sarcoma Virus, as described in Gorman et al., Proc. Natl. Acad.Sci. USA (1982b) 79:6777 and elements derived from human CMV, asdescribed in Boshart et al., Cell (1985) 41:521, such as elementsincluded in the CMV intron A sequence (Chapman et al., Nuc. Acids Res.(1991) 19:3979-3986).

The desired synthetic polypeptide encoding sequences can be cloned intoany number of commercially available vectors to generate expression ofthe polypeptide in an appropriate host system. These systems include,but are not limited to, the following: baculovirus expression (Reilly,P. R., et al., BACULOVIRUS EXPRESSION VECTORS: A LABORATORY MANUAL(1992); Beames, et al., Biotechniques 11:378 (1991); Pharmingen;Clontech, Palo Alto, Calif.)}, vaccinia expression {Earl, P. L., et al.,“Expression of proteins in mammalian cells using vaccinia” In CurrentProtocols in Molecular Biology (F. M. Ausubel, et al. Eds.), GreenePublishing Associates & Wiley Interscience, New York (1991); Moss, B.,et al., U.S. Pat. No. 5,135,855, issued 4 Aug. 1992}, expression inbacteria {Ausubel, F. M., et al., CURRENT PROTOCOLS IN MOLECULARBIOLOGY, John Wiley and Sons, Inc., Media Pa.; Clontech}, expression inyeast {Rosenberg, S. and Tekamp-Olson, P., U.S. Pat. No. RE35,749,issued, Mar. 17, 1998, herein incorporated by reference; Shuster, J. R.,U.S. Pat. No. 5,629,203, issued May 13, 1997, herein incorporated byreference; Gellissen, G., et al., Antonie Van Leeuwenhoek, 62(1-2):79-93(1992); Romanos, M. A., et al., Yeast 8(6):423-488 (1992); Goeddel, D.V., Methods in Enzymology 185 (1990); Guthrie, C., and G. R. Fink,Methods in Enzymology 194 (1991)}, expression in mammalian cells{Clontech; Gibco-BRL, Ground Island, N.Y.; e.g., Chinese hamster ovary(CHO) cell lines (Haynes, J., et al., Nuc. Acid. Res. 11:687-706 (1983);1983, Lau, Y. F., et al., Mol. Cell. Biol. 4:1469-1475 (1984); Kaufman,R. J., “Selection and coamplification of heterologous genes in mammaliancells,” in Methods in Enzymology, vol. 185, pp 537-566. Academic Press,Inc., San Diego Calif. (1991)}, and expression in plant cells {plantcloning vectors, Clontech Laboratories, Inc., Palo Alto, Calif., andPharmacia LKB Biotechnology, Inc., Pistcataway, N.J.; Hood, E., et al.,J. Bacteriol. 168:1291-1301 (1986); Nagel, R., et al., FEMS Microbiol.Lett. 67:325 (1990); An, et al., “Binary Vectors”, and others in PlantMolecular Biology Manual A3:1-19 (1988); Miki, B. L. A., et al., pp.249-65, and others in Plant DNA Infectious Agents (Hohn, T., et al.,eds.) Springer-Verlag, Wien, Austria, (1987); Plant Molecular Biology:Essential Techniques, P. G. Jones and J. M. Sutton, New York, J. Wiley,1997; Miglani, Gurbachan Dictionary of Plant Genetics and MolecularBiology, New York, Food Products Press, 1998; Henry, R. J., PracticalApplications of Plant Molecular Biology, New York, Chapman & Hall,1997}.

Also included in the invention is an expression vector, containingcoding sequences and expression control elements which allow expressionof the coding regions in a suitable host. The control elements generallyinclude a promoter, translation initiation codon, and translation andtranscription termination sequences, and an insertion site forintroducing the insert into the vector. Translational control elementshave been reviewed by M. Kozak (e.g., Kozak, M., Mamm. Genome7(8):563-574, 1996; Kozak, M., Biochimie 76(9):815-821, 1994; Kozak, M.,J Cell Biol 108(2):229-241, 1989; Kozak, M., and Shatkin, A. J., MethodsEnzymol 60:360-375, 1979).

Expression in yeast systems has the advantage of commercial production.Recombinant protein production by vaccinia and CHO cell line have theadvantage of being mammalian expression systems. Further, vaccinia virusexpression has several advantages including the following: (i) its widehost range; (ii) faithful post-transcriptional modification, processing,folding, transport, secretion, and assembly of recombinant proteins;(iii) high level expression of relatively soluble recombinant proteins;and (iv) a large capacity to accommodate foreign DNA.

The recombinantly expressed polypeptides from synthetic HIVpolypeptide-encoding expression cassettes are typically isolated fromlysed cells or culture media. Purification can be carried out by methodsknown in the art including salt fractionation, ion exchangechromatography, gel filtration, size-exclusion chromatography,size-fractionation, and affinity chromatography. Immunoaffinitychromatography can be employed using antibodies generated based on, forexample, HIV antigens.

Advantages of expressing the proteins of the present invention usingmammalian cells include, but are not limited to, the following:well-established protocols for scale-up production; the ability toproduce VLPs; cell lines are suitable to meet good manufacturing process(GMP) standards; culture conditions for mammalian cells are known in theart.

Synthetic HIV 1 polynucleotides are described herein, see, for example,the figures. Various forms of the different embodiments of theinvention, described herein, may be combined.

Exemplary expression assays are set forth in Example 2. Exemplaryconditions for Western Blot analysis are presented in Example 3.

2.3.0 Production of Virus-Like Particles and Use of the Constructs ofthe Present Invention to Create Packaging Cell Lines.

The group-specific antigens (Gag) of human immunodeficiency virus type-1(HIV-1) self-assemble into noninfectious virus-like particles (VLP) thatare released from various eucaryotic cells by budding (reviewed byFreed, E. O., Virology 251:1-15, 1998). The Gag-containing syntheticexpression cassettes of the present invention provide for the productionof HIV-Gag virus-like particles (VLPs) using a variety of different celltypes, including, but not limited to, mammalian cells.

Viral particles can be used as a matrix for the proper presentation ofan antigen entrapped or associated therewith to the immune system of thehost.

2.3.1 VLP Production Using the Synthetic Expression Cassettes of thePresent Invention

The Gag-containing synthetic expression cassettes of the presentinvention may provide superior production of both Gag proteins and VLPs,relative to native Gag coding sequences. Further, electron microscopicevaluation of VLP production can be used to show that free and buddingimmature virus particles of the expected size are produced by cellscontaining the synthetic expression cassettes.

Using the synthetic expression cassettes of the present invention,rather than native Gag coding sequences, for the production ofvirus-like particles provide several advantages. First, VLPs can beproduced in enhanced quantity making isolation and purification of theVLPs easier. Second, VLPs can be produced in a variety of cell typesusing the synthetic expression cassettes, in particular, mammalian celllines can be used for VLP production, for example, CHO cells. Productionusing CHO cells provides (i) VLP formation; (ii) correct myristoylationand budding; (iii) absence of non-Macmillian cell contaminants (e.g.,insect viruses and/or cells); and (iv) ease of purification. Thesynthetic expression cassettes of the present invention are also usefulfor enhanced expression in cell-types other than mammalian cell lines.For example, infection of insect cells with baculovirus vectors encodingthe synthetic expression cassettes results in higher levels of total Gagprotein yield and higher levels of VLP production (relative towild-coding sequences). Further, the final product from insect cellsinfected with the baculovirus-Gag synthetic expression cassettesconsistently contains lower amounts

of contaminating insect proteins than the final product when wild-codingsequences are used.

VLPs can spontaneously form when the particle-forming polypeptide ofinterest is recombinantly expressed in an appropriate host cell. Thus,the VLPs produced using the synthetic expression cassettes of thepresent invention are conveniently prepared using recombinanttechniques. As discussed below, the Gag polypeptide encoding syntheticexpression cassettes of the present invention can include otherpolypeptide coding sequences of interest (for example, HIV protease, HIVpolymerase, Env; synthetic Env). Expression of such synthetic expressioncassettes yields VLPs comprising the Gag polypeptide, as well as, thepolypeptide of interest.

Once coding sequences for the desired particle-forming polypeptides havebeen isolated or synthesized, they can be cloned into any suitablevector or replicon for expression. Numerous cloning vectors are known tothose of skill in the art, and the selection of an appropriate cloningvector is a matter of choice. See, generally, Sambrook et al, supra. Thevector is then used to transform an appropriate host cell. Suitablerecombinant expression systems include, but are not limited to,bacterial, mammalian, baculovirus/insect, vaccinia, Semliki Forest virus(SFV), Alphaviruses (such as, Sindbis, Venezuelan Equine Encephalitis(VEE)), mammalian, yeast and Xenopus expression systems, well known inthe art. Particularly preferred expression systems are mammalian celllines, vaccinia, Sindbis, eucaryotic layered vector initiation systems(e.g., U.S. Pat. No. 6,015,686, U.S. Pat. No. 5,814,482, U.S. Pat. No.6,015,694, U.S. Pat. No. 5,789,245, EP 1029068A2, WO 9918226A2/A3, EP00907746A2, WO 9738087A2, all herein incorporated by reference in theirentireties), insect and yeast systems.

The synthetic DNA fragments for the expression cassettes of the presentinvention, e.g., Pol, Gag, Env, Tat, Rev, Nef, Vif, Vpr, and/or Vpu, maybe cloned into the following eucaryotic expression vectors: pCMVKm2, fortransient expression assays and DNA immunization studies, the pCMVKm2vector is derived from pCMV6a (Chapman et al., Nuc. Acids Res. (1991)19:3979-3986) and comprises a kanamycin selectable marker, a ColE1origin of replication, a CMV promoter enhancer and Intron A, followed byan insertion site for the synthetic sequences described below followedby a polyadenylation signal derived from bovine growth hormone—thepCMVKm2 vector differs from the pCMV-link vector only in that apolylinker site is inserted into pCMVKm2 to generate pCMV-link;pESN2dhfr and pCMVPLEdhfr, for expression in Chinese Hamster Ovary (CHO)cells; and, pAcC13, a shuttle vector for use in the Baculovirusexpression system (pAcC13, is derived from pAcC12 which is described byMunemitsu S., et al., Mol Cell Biol. 10(11):5977-5982, 1990).

Briefly, construction of pCMVPLEdhfr was as follows.

To construct a DHFR cassette, the EMCV IRES (internal ribosome entrysite) leader was PCR-amplified from pCite-4a+(Novagen, Inc., Milwaukee,Wis.) and inserted into pET-23d (Novagen, Inc., Milwaukee, Wis.) as anXba-Nco fragment to give pET-EMCV. The dhfr gene was PCR-amplified frompESN2dhfr to give a product with a Gly-Gly-Gly-Ser spacer in place ofthe translation stop codon and inserted as an Nco-BamH1 fragment to givepET-E-DHFR. Next, the attenuated neo gene was PCR amplified from apSV2Neo (Clontech, Palo Alto, Calif.) derivative and inserted into theunique BamH1 site of pET-E-DHFR to give pET-E-DHFR/Neo_((m2)). Finallythe bovine growth hormone terminator from pCDNA3 (Invitrogen, Inc.,Carlsbad, Calif.) was inserted downstream of the neo gene to givepET-E-DHFR Neo_((m2))BGHt. The EMCV-dhfr/neo selectable marker cassettefragment was prepared by cleavage of pET-E-DHFR/Neo_((m2))BGHt.

In one vector construct the CMV enhancer/promoter plus Intron A wastransferred from pCMV6a (Chapman et al., Nuc. Acids Res. (1991)19:3979-3986) as a HindIII-Sal1 fragment into pUC19 (New EnglandBiolabs, Inc., Beverly, Mass.). The vector backbone of pUC19 was deletedfrom the Nde1 to the Sap1 sites. The above described DHFR cassette wasadded to the construct such that the EMCV IRES followed the CMVpromoter. The vector also contained an ampr gene and an SV40 origin ofreplication.

A number of mammalian cell lines are known in the art and includeimmortal-ized cell lines available from the American Type CultureCollection (A.T.C.C.), such as, but not limited to, Chinese hamsterovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkeykidney cells (COS), as well as others. Similarly, bacterial hosts suchas E. coli, Bacillus subtilis, and Streptococcus spp., will find usewith the present expression constructs. Yeast hosts useful in thepresent invention include inter alia, Saccharomyces cerevisiae, Candidaalbicans, Candida maltosa, Hansenula polymorpha, Kluyveromyces fragilis,Kluyveromyces lactis, Pichia guillerimondii, Pichia pastoris,Schizosaccharomyces pombe and Yarrowia lipolytica. Insect cells for usewith baculovirus expression vectors include, inter alia, Aedes aegypti,Autographa californica, Bombyx mori, Drosophila melanogaster, Spodopterafrugiperda, and Trichoplusia ni. See, e.g., Summers and Smith, TexasAgricultural Experiment Station Bulletin No. 1555 (1987).

Viral vectors can be used for the production of particles in eucaryoticcells, such as those derived from the pox family of viruses, includingvaccinia virus and avian poxvirus. Additionally, a vaccinia basedinfection/transfection system, as described in Tomei et al., J. Virol.(1993) 67:40174026 and Selby et al., J. Gen. Virol. (1993) 74:1103-1113, will also find use with the present invention. In thissystem, cells are first infected in vitro with a vaccinia virusrecombinant that encodes the bacteriophage T7 RNA polymerase. Thispolymerase displays exquisite specificity in that it only transcribestemplates bearing T7 promoters. Following infection, cells aretransfected with the DNA of interest, driven by a T7 promoter. Thepolymerase expressed in the cytoplasm from the vaccinia virusrecombinant transcribes the transfected DNA into RNA which is thentranslated into protein by the host translational machinery.Alternately, T7 can be added as a purified protein or enzyme as in the“Progenitor” system (Studier and Moffatt, J. Mol. Biol. (1986)189:113-130). The method provides for high level, transient, cytoplasmicproduction of large quantities of RNA and its translation product(s).

Depending on the expression system and host selected, the VLPS areproduced by growing host cells transformed by an expression vector underconditions whereby the particle-forming polypeptide is expressed andVLPs can be formed. The selection of the appropriate growth conditionsis within the skill of the art. If the VLPs are formed intracellularly,the cells are then disrupted, using chemical, physical or mechanicalmeans, which lyse the cells yet keep the VLPs substantially intact. Suchmethods are known to those of skill in the art and are described in,e.g., Protein Purification Applications: A Practical Approach, (E. L. V.Harris and S. Angal, Eds., 1990).

The particles are then isolated (or substantially purified) usingmethods that preserve the integrity thereof, such as, by gradientcentrifugation, e.g., cesium chloride (CsCl) sucrose gradients,pelleting and the like (see, e.g., Kirnbauer et al. J. Virol. (1993)67:6929-6936), as well as standard purification techniques including,e.g., ion exchange and gel filtration chromatography.

VLPs produced by cells containing the synthetic expression cassettes ofthe present invention can be used to elicit an immune response whenadministered to a subject. One advantage of the present invention isthat VLPs can be produced by mammalian cells carrying the syntheticexpression cassettes at levels previously not possible. As discussedabove, the VLPs can comprise a variety of antigens in addition to theGag polypeptide (e.g., Gag-protease, Gag-polymerase, Env, synthetic Env,etc.). Purified VLPs, produced using the synthetic expression cassettesof the present invention, can be administered to a vertebrate subject,usually in the form of vaccine compositions. Combination vaccines mayalso be used, where such vaccines contain, for example, an adjuvantsubunit protein (e.g., Env). Administration can take place using theVLPs formulated alone or formulated with other antigens. Further, theVLPs can be administered prior to, concurrent with, or subsequent to,delivery of the synthetic expression cassettes for DNA immunization (seebelow) and/or delivery of other vaccines. Also, the site of VLPadministration may be the same or different as other vaccinecompositions that are being administered. Gene delivery can beaccomplished by a number of methods including, but are not limited to,immunization with DNA, alphavirus vectors, pox virus vectors, andvaccinia virus vectors.

VLP immune-stimulating (or vaccine) compositions can include variousexcipients, adjuvants, carriers, auxiliary substances, modulatingagents, and the like. The immune stimulating compositions will includean amount of the VLP/antigen sufficient to mount an immunologicalresponse. An appropriate effective amount can be determined by one ofskill in the art. Such an amount will fall in a relatively broad rangethat can be determined through routine trials and will generally be anamount on the order of about 0.1 μg to about 1000 μg, more preferablyabout 1 μg to about 300 μg, of VLP/antigen.

A carrier is optionally present which is a molecule that does not itselfinduce the production of antibodies harmful to the individual receivingthe composition. Suitable carriers are typically large, slowlymetabolized macromolecules such as proteins, polysaccharides, polylacticacids, polyglycollic acids, polymeric amino acids, amino acidcopolymers, lipid aggregates (such as oil droplets or liposomes), andinactive virus particles. Examples of particulate carriers include thosederived from polymethyl methacrylate polymers, as well as microparticlesderived from poly(lactides) and poly(lactide-co-glycolides), known asPLG. See, e.g., Jeffery et al., Pharm. Res. (1993) 10:362-368; McGee JP, et al., J Microencapsul. 14(2): 197-210, 1997; O'Hagan D T, et al.,Vaccine 11(2): 149-54, 1993. Such carriers are well known to those ofordinary skill in the art. Additionally, these carriers may function asimmunostimulating agents (“adjuvants”). Furthermore, the antigen may beconjugated to a bacterial toxoid, such as toxoid from diphtheria,tetanus, cholera, etc., as well as toxins derived from E. coli.

Adjuvants may also be used to enhance the effectiveness of thecompositions. Such adjuvants include, but are not limited to: (1)aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate,aluminum sulfate, etc.; (2) oil-in-water emulsion formulations (with orwithout other specific immunostimulating agents such as muramyl peptides(see below) or bacterial cell wall components), such as for example (a)MF59 (International Publication No. WO 90/14837), containing 5%Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionally containing variousamounts of MTP-PE (see below), although not required) formulated intosubmicron particles using a microfluidizer such as Model 110Ymicrofluidizer (Microfluidics, Newton, Mass.), (b) SAF, containing 10%Squalane, 0.4% Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP(see below) either microfluidized into a submicron emulsion or vortexedto generate a larger particle size emulsion, and (c) Ribi™ adjuvantsystem (RAS), (Ribi Immunochem, Hamilton, Mont.) containing 2% Squalene,0.2% Tween 80, and one or more bacterial cell wall components from thegroup consisting of monophosphorylipid A (MPL), trehalose dimycolate(TDM), and cell wall skeleton (CWS), preferably MPL+CWS (Detox™); (3)saponin adjuvants, such as Stimulon™ (Cambridge Bioscience, Worcester,Mass.) may be used or particle generated therefrom such as ISCOMs(immunostimulating complexes); (4) Complete Freunds Adjuvant (CFA) andIncomplete Freunds Adjuvant (IFA); (5) cytokines, such as interleukins(IL-1, IL-2, etc.), macrophage colony stimulating factor (M-CSF), tumornecrosis factor (TNF), etc.; (6) oligonucleotides or polymeric moleculesencoding immunostimulatory CpG motifs (Davis, H. L., et al., J.Immunology 160:870-876, 1998; Sato, Y. et al., Science 273:352-354,1996) or complexes of antigens/oligonucleotides {Polymeric moleculesinclude double and single stranded RNA and DNA, and backbonemodifications thereof, for example, methylphosphonate linkages; or (7)detoxified mutants of a bacterial ADP-ribosylating toxin such as acholera toxin (CT), a pertussis toxin (PT), or an E. coli heat-labiletoxin (LT), particularly LT-K63 (where lysine is substituted for thewild-type amino acid at position 63) LT-R72 (where arginine issubstituted for the wild-type amino acid at position 72), CT-S109 (whereserine is substituted for the wild-type amino acid at position 109), andPT-K9/G129 (where lysine is substituted for the wild-type amino acid atposition 9 and glycine substituted at position 129) (see, e.g.,International Publication Nos. W093/13202 and W092/19265); and (8) othersubstances that act as immunostimulating agents to enhance theeffectiveness of the composition. Further, such polymeric moleculesinclude alternative polymer backbone structures such as, but not limitedto, polyvinyl backbones (Pitha, Biochem Biophys Acta, 204:39, 1970a;Pitha, Biopolymers, 9:965, 1970b), and morpholino backbones (Summerton,J., et al., U.S. Pat. No. 5,142,047, issued Aug. 25, 1992; Summerton,J., et al., U.S. Pat. No. 5,185,444 issued Feb. 9, 1993). A variety ofother charged and uncharged polynucleotide analogs have been reported.Numerous backbone modifications are known in the art, including, but notlimited to, uncharged linkages (e.g., methyl phosphonates,phosphotriesters, phosphoamidates, and carbamates) and charged linkages(e.g., phosphorothioates and phosphorodithioates).}; and (7) othersubstances that act as immunostimulating agents to enhance theeffectiveness of the VLP immune-stimulating (or vaccine) composition.Alum, CpG oligonucleotides, and MF59 are preferred.

Muramyl peptides include, but are not limited to,N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acteyl-normuramyl-Lalanyl-D-isogluatme (nor-MDP),N-acetylmuramyl-L-alanyl-D-isogluatminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-huydroxyphosphoryloxy)-ethylamine(MTP-PE), etc.

Dosage treatment with the VLP composition may be a single dose scheduleor a multiple dose schedule. A multiple dose schedule is one in which aprimary course of vaccination may be with 1-10 separate doses, followedby other doses given at subsequent time intervals, chosen to maintainand/or reinforce the immune response, for example at 1-4 months for asecond dose, and if needed, a subsequent dose(s) after several months.The dosage regimen will also, at least in part, be determined by theneed of the subject and be dependent on the judgment of thepractitioner.

If prevention of disease is desired, the antigen carrying VLPs aregenerally administered prior to primary infection with the pathogen ofinterest. If treatment is desired, e.g., the reduction of symptoms orrecurrences, the VLP compositions are generally administered subsequentto primary infection.

2.3.2 Using the Synthetic Expression Cassettes of the Present Inventionto Create Packaging Cell Lines

A number of viral based systems have been developed for use as genetransfer vectors for mammalian host cells. For example, retroviruses (inparticular, lentiviral vectors) provide a convenient platform for genedelivery systems. A coding sequence of interest (for example, a sequenceuseful for gene therapy applications) can be inserted into a genedelivery vector and packaged in retroviral particles using techniquesknown in the art. Recombinant virus can then be isolated and deliveredto cells of the subject either in vivo or ex vivo. A number ofretroviral systems have been described, including, for example, thefollowing: (U.S. Pat. No. 5,219,740; Miller et al. (1989) BioTechniques7:980; Miller, A. D. (1990) Human Gene Therapy 1:5; Scarpa et al. (1991)Virology 180:849; Burns et al. (1993) Proc. Natl. Acad. Sci. USA90:8033; Boris-Lawrie et al. (1993) Cur. Opin. Genet. Develop. 3:102; GB2200651; EP 0415731; EP 0345242; WO 89/02468; WO 89/05349; WO 89/09271;WO 90/02806; WO 90/07936; WO 90/07936; WO 94/03622; WO 93/25698; WO93/25234; WO 93/11230; WO 93/10218; WO 91/02805; in U.S. Pat. No.5,219,740; U.S. Pat. No. 4,405,712; U.S. Pat. No. 4,861,719; U.S. Pat.No. 4,980,289 and U.S. Pat. No. 4,777,127; in U.S. Ser. No. 07/800,921;and in Vile (1993) Cancer Res 53:3860-3864; Vile (1993) Cancer Res53:962-967; Ram (1993) Cancer Res 53:83-88; Takamiya (1992) J NeurosciRes 33:493-503; Baba (1993) J Neurosurg 79:729-735; Mann (1983) Cell33:153; Cane (1984) Proc Natl Acad Sci USA 81; 6349; and Miller (1990)Human Gene Therapy 1.

In other embodiments, gene transfer vectors can be constructed to encodea cytokine or other immunomodulatory molecule. For example, nucleic acidsequences encoding native IL-2 and gamma-interferon can be obtained asdescribed in U.S. Pat. Nos. 4,738,927 and 5,326,859, respectively, whileuseful muteins of these proteins can be obtained as described in U.S.Pat. No. 4,853,332. Nucleic acid sequences encoding the short and longforms of mCSF can be obtained as described in U.S. Pat. Nos. 4,847,201and 4,879,227, respectively. In particular aspects of the invention,retroviral vectors expressing cytokine or immunomodulatory genes can beproduced as described herein (for example, employing the packaging celllines of the present invention) and in International Application No. PCTUS 94/02951, entitled “Compositions and Methods for CancerImmunotherapy.”

Examples of suitable immunomodulatory molecules for use herein includethe following: IL-1 and IL-2 (Karupiah et al. (1990) J. Immunology144:290-298, Weber et al. (1987) J. Exp. Med. 166:1716-1733, Gansbacheret al. (1990) J. Exp. Med. 172:1217-1224, and U.S. Pat. No. 4,738,927);IL-3 and IL-4 (Tepper et al. (1989) Cell 57:503-512, Golumbek et al.(1991) Science 254:713-716, and U.S. Pat. No. 5,017,691); IL-5 and IL-6(Brakenhof et al. (1987) J. Immunol. 139:4116-4121, and InternationalPublication No. WO 90/06370); IL-7 (U.S. Pat. No. 4,965,195); IL-8,IL-9, IL-10, IL-11, IL-12, and IL-13 (Cytokine Bulletin, Summer 1994);IL-14 and IL-15; alpha interferon (Finter et al. (1991) Drugs42:749-765, U.S. Pat. Nos. 4,892,743 and 4,966,843, InternationalPublication No. WO 85/02862, Nagata et al. (1980) Nature 284:316-320,Familletti et al. (1981) Methods in Enz. 78:387-394, Twu et al. (1989)Proc. Natl. Acad. Sci. USA 86:2046-2050, and Faktor et al. (1990)Oncogene 5:867-872); beta-interferon (Seif et al. (1991) J. Virol.65:664-671); gamma-interferons (Radford et al. (1991) The AmericanSociety of Hepatology 20082015, Watanabe et al. (1989) Proc. Natl. Acad.Sci. USA 86:9456-9460, Gansbacher et al. (1990) Cancer Research50:7820-7825, Maio et al. (1989) Can. Immunol. Immunother. 30:34-42, andU.S. Pat. Nos. 4,762,791 and 4,727,138); G-CSF (U.S. Pat. Nos. 4,999,291and 4,810,643); GM-CSF (International Publication No. WO 85/04188).

Immunomodulatory factors may also be agonists, antagonists, or ligandsfor these molecules. For example, soluble forms of receptors can oftenbehave as antagonists for these types of factors, as can mutated formsof the factors themselves.

Nucleic acid molecules that encode the above-described substances, aswell as other nucleic acid molecules that are advantageous for usewithin the present invention, may be readily obtained from a variety ofsources, including, for example, depositories such as the American TypeCulture Collection, or from commercial sources such as BritishBio-Technology Limited (Cowley, Oxford England). Representative examplesinclude BBG 12 (containing the GM-CSF gene coding for the mature proteinof 127 amino acids), BBG 6 (which contains sequences encoding gammainterferon), A.T.C.C. Deposit No. 39656 (which contains sequencesencoding TNF), A.T.C.C. Deposit No. 20663 (which contains sequencesencoding alpha-interferon), A.T.C.C. Deposit Nos. 31902, 31902 and 39517(which contain sequences encoding beta-interferon), A.T.C.C. Deposit No.67024 (which contains a sequence which encodes Interleukin-1b), A.T.C.C.Deposit Nos. 39405, 39452, 39516, 39626 and 39673 (which containsequences encoding Interleukin-2), A.T.C.C. Deposit Nos. 59399, 59398,and 67326 (which contain sequences encoding Interleukin-3), A.T.C.C.Deposit No. 57592 (which contains sequences encoding Interleukin-4),A.T.C.C. Deposit Nos. 59394 and 59395 (which contain sequences encodingInterleukin-5), and A.T.C.C. Deposit No. 67153 (which contains sequencesencoding Interleukin-6).

Plasmids containing cytokine genes or immunomodulatory genes(International Publication Nos. WO 94/02951 and WO 96/21015, both ofwhich are incorporated by reference in their entirety) can be digestedwith appropriate restriction enzymes; and DNA fragments containing theparticular gene of interest can be inserted into a gene transfer vectorusing standard molecular biology techniques. (See, e.g., Sambrook etal., supra., or Ausubel et al. (eds) Current Protocols in MolecularBiology, Greene Publishing and Wiley-Interscience).

Polynucleotide sequences coding for the above-described molecules can beobtained using recombinant methods, such as by screening cDNA andgenomic libraries from cells expressing the gene, or by deriving thegene from a vector known to include the same. For example, plasmidswhich contain sequences that encode altered cellular products may beobtained from a depository such as the A.T.C.C., or from commercialsources. Plasmids containing the nucleotide sequences of interest can bedigested with appropriate restriction enzymes, and DNA fragmentscontaining the nucleotide sequences can be inserted into a gene transfervector using standard molecular biology techniques.

Alternatively, cDNA sequences for use with the present invention may beobtained from cells which express or contain the sequences, usingstandard techniques, such as phenol extraction and PCR of cDNA orgenomic DNA. See, e.g., Sambrook et al., supra, for a description oftechniques used to obtain and isolate DNA. Briefly, mRNA from a cellwhich expresses the gene of interest can be reverse transcribed withreverse transcriptase using oligo-dT or random primers. The singlestranded cDNA may then be amplified by PCR (see U.S. Pat. Nos.4,683,202, 4,683,195 and 4,800,159, see also PCR Technology: Principlesand Applications for DNA Amplification, Erlich (ed.), Stockton Press,1989)) using oligonucleotide primers complementary to sequences oneither side of desired sequences.

The nucleotide sequence of interest can also be produced synthetically,rather than cloned, using a DNA synthesizer (e.g., an Applied BiosystemsModel 392 DNA Synthesizer, available from ABI, Foster City, Calif.). Thenucleotide sequence can be designed with the appropriate codons for theexpression product desired. The complete sequence is assembled fromoverlapping oligonucleotides prepared by standard methods and assembledinto a complete coding sequence. See, e.g., Edge (1981) Nature 292:756;Nambair et al. (1984) Science 223:1299; Jay et al. (1984) J. Biol. Chem.259:6311.

The synthetic expression cassettes of the present invention can beemployed in the construction of packaging cell lines for use withretroviral vectors.

One type of retrovirus, the murine leukemia virus, or “MLV”, has beenwidely utilized for gene therapy applications (see generally Mann et al.(Cell 33:153, 1993), Cane and Mulligan (Proc, Nat'l Acad. Sci. USA81:6349, 1984), and Miller et al., Human Gene Therapy 1:5-14, 1990.

Lentiviral vectors typically, comprise a 5′ lentiviral LTR, a tRNAbinding site, a packaging signal, a promoter operably linked to one ormore genes of interest, an origin of second strand DNA synthesis and a3′ lentiviral LTR, wherein the lentiviral vector contains a nucleartransport element. The nuclear transport element may be located eitherupstream (5′) or downstream (3′) of a coding sequence of interest (forexample, a synthetic Gag or Env expression cassette of the presentinvention). Within certain embodiments, the nuclear transport element isnot RRE. Within one embodiment the packaging signal is an extendedpackaging signal. Within other embodiments the promoter is a tissuespecific promoter, or, alternatively, a promoter such as CMV. Withinother embodiments, the lentiviral vector further comprises an internalribosome entry site.

A wide variety of lentiviruses may be utilized within the context of thepresent invention, including for example, lentiviruses selected from thegroup consisting of HIV, HIV-1, HIV-2, FIV and SIV.

Within yet another aspect of the invention, host cells (e.g., packagingcell lines) are provided which contain any of the expression cassettesdescribed herein. For example, within one aspect packaging cell line areprovided comprising an expression cassette that comprises a sequenceencoding synthetic Gag-polymerase, and a nuclear transport element,wherein the promoter is operably linked to the sequence encodingGag-polymerase. Packaging cell lines may further comprise a promoter anda sequence encoding tat, rev, or an envelope, wherein the promoter isoperably linked to the sequence encoding tat, rev, Env or sequencesencoding modified versions of these proteins. The packaging cell linemay further comprise a sequence encoding any one or more of other HIVgene encoding sequences.

In one embodiment, the expression cassette (carrying, for example, thesynthetic Gag-polymerase) is stably integrated. The packaging cell line,upon introduction of a lentiviral vector, typically produces particles.The promoter regulating expression of the synthetic expression cassettemay be inducible. Typically, the packaging cell line, upon introductionof a lentiviral vector, produces particles that are essentially free ofreplication competent virus.

Packaging cell lines are provided comprising an expression cassettewhich directs the expression of a synthetic Gag-polymerase gene orcomprising an expression cassette which directs the expression of asynthetic Env genes described herein. (See, also, Andre, S., et al.,Journal of Virology 72(2):1497-1503, 1998; Haas, J., et al., CurrentBiology 6(3):315-324, 1996) for a description of other modified Envsequences). A lentiviral vector is introduced into the packaging cellline to produce a vector producing cell line.

As noted above, lentiviral vectors can be designed to carry or express aselected gene(s) or sequences of interest. Lentiviral vectors may bereadily constructed from a wide variety of lentiviruses (see RNA TumorViruses, Second Edition, Cold Spring Harbor Laboratory, 1985).Representative examples of lentiviruses included HIV, HIV-1, HIV-2, FIVand SIV. Such lentiviruses may either be obtained from patient isolates,or, more preferably, from depositories or collections such as theAmerican Type Culture Collection, or isolated from known sources usingavailable techniques.

Portions of the lentiviral gene delivery vectors (or vehicles) may bederived from different viruses. For example, in a given recombinantlentiviral vector, LTRs may be derived from an HIV, a packaging signalfrom SIV, and an origin of second strand synthesis from HrV-2.Lentiviral vector constructs may comprise a 5′ lentiviral LTR, a tRNAbinding site, a packaging signal, one or more heterologous sequences, anorigin of second strand DNA synthesis and a 3′ LTR, wherein saidlentiviral vector contains a nuclear transport element that is not RRE.

Briefly, Long Terminal Repeats (“LTRs”) are subdivided into threeelements, designated U5, R and U3. These elements contain a variety ofsignals which are responsible for the biological activity of aretrovirus, including for example, promoter and enhancer elements whichare located within U3. LTRs may be readily identified in the provirus(integrated DNA form) due to their precise duplication at either end ofthe genome. As utilized herein, a 5′ LTR should be understood to includea 5′ promoter element and sufficient LTR sequence to allow reversetranscription and integration of the DNA form of the vector. The 3′ LTRshould be understood to include a polyadenylation signal, and sufficientLTR sequence to allow reverse transcription and integration of the DNAform of the vector.

The tRNA binding site and origin of second strand DNA synthesis are alsoimportant for a retrovirus to be biologically active, and may be readilyidentified by one of skill in the art. For example, retroviral tRNAbinds to a tRNA binding site by Watson-Crick base pairing, and iscarried with the retrovirus genome into a viral particle. The tRNA isthen utilized as a primer for DNA synthesis by reverse transcriptase.The tRNA binding site may be readily identified based upon its locationjust downstream from the 5′LTR. Similarly, the origin of second strandDNA synthesis is, as its name implies, important for the second strandDNA synthesis of a retrovirus. This region, which is also referred to asthe poly-purine tract, is located just upstream of the 3′LTR.

In addition to a 5′ and 3′ LTR, tRNA binding site, and origin of secondstrand DNA synthesis, recombinant retroviral vector constructs may alsocomprise a packaging signal, as well as one or more genes or codingsequences of interest. In addition, the lentiviral vectors have anuclear transport element which, in preferred embodiments is not RRE.Representative examples of suitable nuclear transport elements includethe element in Rous sarcoma virus (Ogert, et al., J. Virol. 70,3834-3843, 1996), the element in Rous sarcoma virus (Liu & Mertz, Genes& Dev., 9, 1766-1789, 1995) and the element in the genome of simianretrovirus type I (Zolotukhin, et al., J. Virol. 68, 7944-7952, 1994).Other potential elements include the elements in the histone gene(Kedes, Annu. Rev. Biochem. 48, 837-870, 1970), the α-interferon gene(Nagata et al., Nature 287, 401-408, 1980), the β-adrenergic receptorgene (Koilka, et al., Nature 329, 75-79, 1987), and the c-Jun gene(Hattorie, et al., Proc. Natl. Acad. Sci. USA 85, 9148-9152, 1988).

Recombinant lentiviral vector constructs typically lack bothGag-polymerase and Env coding sequences. Recombinant lentiviral vectortypically contain less than 20, preferably 15, more preferably 10, andmost preferably 8 consecutive nucleotides found in Gag-polymerase andEnv genes. One advantage of the present invention is that the syntheticGag-polymerase expression cassettes, which can be used to constructpackaging cell lines for the recombinant retroviral vector constructs,have little homology to wild-type Gag-polymerase sequences and thusconsiderably reduce or eliminate the possibility of homologousrecombination between the synthetic and wild-type sequences.

Lentiviral vectors may also include tissue-specific promoters to driveexpression of one or more genes or sequences of interest.

Lentiviral vector constructs may be generated such that more than onegene of interest is expressed. This may be accomplished through the useof di- or oligo-cistronic cassettes (e.g., where the coding regions areseparated by 80 nucleotides or less, see generally Levin et al., Gene108:167-174, 1991), or through the use of Internal Ribosome Entry Sites(“IRES”).

Packaging cell lines suitable for use with the above describedrecombinant retroviral vector constructs may be readily prepared giventhe disclosure provided herein. Briefly, the parent cell line from whichthe packaging cell line is derived can be selected from a variety ofmammalian cell lines, including for example, 293, RD, COS-7, CHO, BHK,VERO, HT1080, and myeloma cells.

After selection of a suitable host cell for the generation of apackaging cell line, one or more expression cassettes are introducedinto the cell line in order to complement or supply in trans componentsof the vector which have been deleted.

Representative examples of suitable synthetic HIV polynucleotidesequences have been described herein for use in expression cassettes ofthe present invention. As described above, the native and/or syntheticcoding sequences may also be utilized in these expression cassettes.

Utilizing the above-described expression cassettes, a wide variety ofpackaging cell lines can be generated. For example, within one aspectpackaging cell line are provided comprising an expression cassette thatcomprises a sequence encoding synthetic Gag-polymerase, and a nucleartransport element, wherein the promoter is operably linked to thesequence encoding Gag-polymerase. Within other aspects, packaging celllines are provided comprising a promoter and a sequence encoding tat,rev, Env, or other HIV antigens or epitopes derived therefrom, whereinthe promoter is operably linked to the sequence encoding tat, rev, Env,or the HIV antigen or epitope. Within further embodiments, the packagingcell line may comprise a sequence encoding any one or more of tat, rev,nef, vif, vpu or vpr. For example, the packaging cell line may containonly tat, rev, nef, vif, vpu, or vpr alone, tat rev and nef, nef andvif, nef and vpu, nef and vpr, vif and vpu, vif and vpr, vpu and vpr,nef vif and vpu, nef vif and vpr, nef vpu and vpr, vif vpu and vpr, allfour of nef, vif, vpu, and vpr, etc.

In one embodiment, the expression cassette is stably integrated. Withinanother embodiment, the packaging cell line, upon introduction of alentiviral vector, produces particles. Within further embodiments thepromoter is inducible. Within certain preferred embodiments of theinvention, the packaging cell line, upon introduction of a lentiviralvector, produces particles that are free of replication competent virus.

The synthetic cassettes containing modified coding sequences aretransfected into a selected cell line. Transfected cells are selectedthat (i) carry, typically, integrated, stable copies of the HIV codingsequences, and (ii) are expressing acceptable levels of thesepolypeptides (expression can be evaluated by methods known in the priorart in view of the teachings of the present disclosure). The ability ofthe cell line to produce VLPs may also be verified.

A sequence of interest is constructed into a suitable viral vector asdiscussed above. This defective virus is then transfected into thepackaging cell line. The packaging cell line provides the viralfunctions necessary for producing virus-like particles into which thedefective viral genome, containing the sequence of interest, arepackaged. These VLPs are then isolated and can be used, for example, ingene delivery or gene therapy.

Further, such packaging cell lines can also be used to produce VLPsalone, which can, for example, be used as adjuvants for administrationwith other antigens or in vaccine compositions. Also, co-expression of aselected sequence of interest encoding a polypeptide (for example, anantigen) in the packaging cell line can also result in the entrapmentand/or association of the selected polypeptide in/with the VLPs.

Various forms of the different embodiments of the present invention(e.g., synthetic constructs) may be combined.

2.4.0 DNA Immunization and Gene Delivery

A variety of HIV polypeptide antigens, particularly HIV antigens, can beused in the practice of the present invention. HIV antigens can beincluded in DNA immunization constructs containing, for example, asynthetic Env expression cassettes, a synthetic Gag expression cassette,a synthetic pol-derived polypeptide expression cassette, a syntheticexpression cassette comprising sequences encoding one or more accessoryor regulatory genes (e.g., tat, rev, nef, vif, vpu, vpr), and/or asynthetic Gag expression cassette fused in-frame to a coding sequencefor the polypeptide antigen (synthetic or wild-type), where expressionof the construct results in VLPs presenting the antigen of interest.

HIV antigens of particular interest to be used in the practice of thepresent invention include pol, tat, rev, nef, vif, vpu, vpr, and otherHIV-1 (also known as HTLV-III, LAV, ARV, etc.) antigens or epitopesderived therefrom, including, but not limited to, antigens such asgp120, gp41, gp160 (both native and modified); Gag; and pol from avariety of isolates including, but not limited to, HIV_(IIIb),HIV_(SF2), HIV-1_(SF162), HIV-1_(SFI170), HIV_(LAV), HIV_(LAI),HIV_(MN), HIV-1_(CM235), HIV-1_(US4), other HIV-1 strains from diversesubtypes (e.g., subtypes, A through G, and O), HIV-2 strains and diversesubtypes (e.g., HIV-2_(UC1), and HIV-2_(UC2)). See, e.g., Myers, et al.,Los Alamos Database, Los Alamos National Laboratory, Los Alamos, N.Mex.; Myers, et al., Human Retroviruses and Aids, 1990, Los Alamos, N.Mex.: Los Alamos National Laboratory. These antigens may be synthetic(as described herein) or wild-type.

To evaluate efficacy, DNA immunization using synthetic expressioncassettes of the present invention can be performed, for example, asfollows. Mice are immunized with a tat/rev/nef synthetic expressioncassette. Other mice are immunized with a tat/rev/nef wild typeexpression cassette. Mouse immunizations with plasmid-DNAs typicallyshow that the synthetic expression cassettes provide a clear improvementof immunogenicity relative to the native expression cassettes. Also, asecond boost immunization will induce a secondary immune response, forexample, after approximately two weeks. Further, the results of CTLassays typically show increased potency of synthetic expressioncassettes for induction of cytotoxic T-lymphocyte (CTL) responses by DNAimmunization.

Exemplary primate studies directed at the evaluation of neutralizingantibodies and cellular immune responses against HIV are describedbelow.

It is readily apparent that the subject invention can be used to mountan immune response to a wide variety of antigens and hence to treat orprevent infection, particularly HIV infection.

2.4.1 Delivery of the Synthetic Expression Cassettes of the PresentInvention

Polynucleotide sequences coding for the above-described molecules can beobtained using recombinant methods, such as by screening cDNA andgenomic libraries from cells expressing the gene, or by deriving thegene from a vector known to include the same. Furthermore, the desiredgene can be isolated directly from cells and tissues containing thesame, using standard techniques, such as phenol extraction and PCR ofcDNA or genomic DNA. See, e.g., Sambrook et al., supra, for adescription of techniques used to obtain and isolate DNA. The gene ofinterest can also be produced synthetically, rather than cloned. Thenucleotide sequence can be designed with the appropriate codons for theparticular amino acid sequence desired. In general, one will selectpreferred codons for the intended host in which the sequence will beexpressed. The complete sequence is assembled from overlappingoligonucleotides prepared by standard methods and assembled into acomplete coding sequence. See, e.g., Edge, Nature (1981) 292:756;Nambair et al., Science (1984) 223:1299; Jay et al., J. Biol. Chem.(1984) 259:6311; Stemmer, W. P. C., (1995) Gene 164:49-53.

Next, the gene sequence encoding the desired antigen can be insertedinto a vector containing a synthetic expression cassette of the presentinvention. In one embodiment, polynucleotides encoding selected antigensare separately cloned into expression vectors (e.g., Env-codingpolynucleotide in a first vector, Gag-coding polynucleotide in a secondvector, Pol-derived polypeptide-coding polynucleotide in a third vector,tat-, rev-, nef-, vif-, vpu-, vpr-coding polynucleotides in furthervectors, etc.). In certain embodiments, the antigen is inserted into oradjacent a synthetic Gag coding sequence such that when the combinedsequence is expressed it results in the production of VLPs comprisingthe Gag polypeptide and the antigen of interest, e.g., Env (native ormodified) or other antigen(s) (native or modified) derived from HIV.Insertions can be made within the coding sequence or at either end ofthe coding sequence (5′, amino terminus of the expressed Gagpolypeptide; or 3′, carboxy terminus of the expressed Gag polypeptide)(Wagner, R., et al., Arch Virol. 127:117-137, 1992; Wagner, R., et al.,Virology 200:162-175, 1994; Wu, X., et al., J. Virol. 69(6):3389-3398,1995; Wang, C-T., et al., Virology 200:524-534, 1994; Chazal, N., etal., Virology 68(1): 111-122, 1994; Griffiths, J. C., et al., J. Virol.67(6):3191-3198, 1993; Reicin, A. S., et al., J. Virol. 69(2):642-650,1995).

Up to 50% of the coding sequences of p55Gag can be deleted withoutaffecting the assembly to virus-like particles and expression efficiency(Borsetti, A., et al, J. Virol. 72(11):9313-9317, 1998; Garnier, L., etal., J Virol 72(6):4667-4677, 1998; Zhang, Y., et al., J Virol 72(3):1782-1789, 1998; Wang, C., et al., J Virol 72(10): 7950-7959, 1998). Inone embodiment of the present invention, immunogenicity of the highlevel expressing synthetic Gag expression cassettes can be increased bythe insertion of different structural or non-HIV antigens, multi-epitopecassettes, or cytokine sequences into deleted regions of Gag sequence.Such deletions may be generated following the teachings of the presentinvention and information available to one of ordinary skill in the art.One possible advantage of this approach, relative to using full-lengthsequences fused to heterologous polypeptides, can be higherexpression/secretion efficiency of the expression product.

When sequences are added to the amino terminal end of Gag, thepolynucleotide can contain coding sequences at the 5′ end that encode asignal for addition of a myristic moiety to the Gag-containingpolypeptide (e.g., sequences that encode Met-Gly).

The ability of Gag-containing polypeptide constructs to form VLPs can beempirically determined following the teachings of the presentspecification.

The synthetic expression cassettes can also include control elementsoperably linked to the coding sequence, which allow for the expressionof the gene in vivo in the subject species. For example, typicalpromoters for mammalian cell expression include the SV40 early promoter,a CMV promoter such as the CMV immediate early promoter, the mousemammary tumor virus LTR promoter, the adenovirus major late promoter (AdMLP), and the herpes simplex virus promoter, among others. Othernonviral promoters, such as a promoter derived from the murinemetallothionein gene, will also find use for mammalian expression.Typically, transcription termination and polyadenylation sequences willalso be present, located 3′ to the translation stop codon. Preferably, asequence for optimization of initiation of translation, located 5′ tothe coding sequence, is also present. Examples of transcriptionterminator/polyadenylation signals include those derived from SV40, asdescribed in Sambrook et al., supra, as well as a bovine growth hormoneterminator sequence.

Enhancer elements may also be used herein to increase expression levelsof the mammalian constructs. Examples include the SV40 early geneenhancer, as described in Dijkema et al., EMBO J. (1985) 4:761, theenhancer/promoter derived from the long terminal repeat (LTR) of theRous Sarcoma Virus, as described in Gorman et al., Proc. Natl. Acad.Sci. USA (1982b) 79:6777 and elements derived from human CMV, asdescribed in Boshart et al., Cell (1985) 41:521, such as elementsincluded in the CMV intron A sequence.

Furthermore, plasmids can be constructed which include a chimericantigen-coding gene sequences, encoding, e.g., multipleantigens/epitopes of interest, for example derived from more than oneviral isolate.

Typically the antigen coding sequences precede or follow the syntheticcoding sequence and the chimeric transcription unit will have a singleopen reading frame encoding both the antigen of interest and thesynthetic coding sequences. Alternatively, multi-cistronic cassettes(e.g., bi-cistronic cassettes) can be constructed allowing expression ofmultiple antigens from a single mRNA using the EMCV IRES, or the like(Example 7).

In one embodiment of the present invention, a nucleic acid immunizingcomposition may comprise, for example, the following: a first expressionvector comprising a Gag expression cassette, a second vector comprisingan Env expression cassette, and a third expression vector comprising aPol expression cassette, or one or more coding region of Pol (e.g.,Prot, RT, RNase, Int), wherein further antigen coding sequences may beassociated with the Pol expression, such antigens may be obtained, forexample, from accessory genes (e.g., vpr, vpu, vif), regulatory genes(e.g., nef, tat, rev), or portions of the Pol sequences (e.g., Prot, RT,RNase, Int)). In another embodiment, a nucleic acid immunizingcomposition may comprise, for example, an expression cassette comprisingany of the synthetic polynucleotide sequences of the present invention.In another embodiment, a nucleic acid immunizing composition maycomprise, for example, an expression cassette comprising codingsequences for a number of HIV genes (or sequences derived from suchgenes) wherein the coding sequences are in-frame and under the controlof a single promoter, for example, Gag-Env constructs, Tat-Rev-Nefconstructs, P2Pol-tat-rev-nef constructs, etc. The synthetic codingsequences of the present invention may be combined in any number ofcombinations depending on the coding sequence products (i.e., HIVpolypeptides) to which, for example, an immunological response isdesired to be raised. In yet another embodiment, synthetic codingsequences for multiple HIV-derived polypeptides may be constructed intoa polycistronic message under the control of a single promoter whereinIRES are placed adjacent the coding sequence for each encodedpolypeptide.

Once complete, the constructs are used for nucleic acid immunizationusing standard gene delivery protocols. Methods for gene delivery areknown in the art. See, e.g., U.S. Pat. Nos. 5,399,346, 5,580,859,5,589,466. Genes can be delivered either directly to the vertebratesubject or, alternatively, delivered ex vivo, to cells derived from thesubject and the cells reimplanted in the subject.

The amount of DNA administered to the subject may vary depending on theantigens and/or delivery protocol. Thus, in certain embodiments, theamount of DNA used per administration (e.g., immunization) may vary fromnanogram to microgram to milligram amounts of DNA, for example, asdescribed in the Examples where the dose given is between about 2nanograms to 20 micrograms or between about 2 nanograms and 10milligrams. Further, as described below and shown in the Examples,multiple administrations of DNA (and/or protein) are contemplated.

A number of viral based systems have been developed for gene transferinto mammalian cells. For example, retroviruses provide a convenientplatform for gene delivery systems. Selected sequences can be insertedinto a vector and packaged in retroviral particles using techniquesknown in the art. The recombinant virus can then be isolated anddelivered to cells of the subject either in vivo or ex vivo. A number ofretroviral systems have been described (U.S. Pat. No. 5,219,740; Millerand Rosman, BioTechniques (1989) 7:980-990; Miller, A. D., Human GeneTherapy (1990) 1:5-14; Scarpa et al., Virology (1991) 180:849-852; Burnset al., Proc. Natl. Acad. Sci. USA (1993) 90:8033-8037; and Boris-Lawrieand Temin, Cur. Opin. Genet. Develop. (1993) 3:102-109.

A number of adenovirus vectors have also been described. Unlikeretroviruses which integrate into the host genome, adenoviruses persistextrachromosomally thus minimizing the risks associated with insertionalmutagenesis (Haj-Ahmad and Graham, J. Virol. (1986) 57:267-274; Bett etal., J. Virol. (1993) 67:5911-5921; Mittereder et al., Human GeneTherapy (1994) 5:717-729; Seth et al., J. Virol. (1994) 68:933-940; Barret al., Gene Therapy (1994) 1:51-58; Berkner, K. L. BioTechniques (1988)6:616-629; and Rich et al., Human Gene Therapy (1993) 4:461-476).

Additionally, various adeno-associated virus (AAV) vector systems havebeen developed for gene delivery. AAV vectors can be readily constructedusing techniques well known in the art. See, e.g., U.S. Pat. Nos.5,173,414 and 5,139,941; International Publication Nos. WO 92/01070(published 23 Jan. 1992) and WO 93/03769 (published 4 Mar. 1993);Lebkowski et al., Molec. Cell. Biol. (1988) 8:3988-3996; Vincent et al.,Vaccines 90 (1990) (Cold Spring Harbor Laboratory Press); Carter, B. J.Current Opinion in Biotechnology (1992) 3:533-539; Muzyczka, N. CurrentTopics in Microbiol. and Immunol. (1992) 158:97-129; Kotin, R. M. HumanGene Therapy (1994) 5:793-801; Shelling and Smith, Gene Therapy (1994)1:165-169; and Zhou et al., J. Exp. Med. (1994) 179:1867-1875.

Another vector system useful for delivering the polynucleotides of thepresent invention is the enterically administered recombinant poxvirusvaccines described by Small, Jr., P. A., et al. (U.S. Pat. No.5,676,950, issued Oct. 14, 1997, herein incorporated by reference).

Additional viral vectors which will find use for delivering the nucleicacid molecules encoding the antigens of interest include those derivedfrom the pox family of viruses, including vaccinia virus and avianpoxvirus. By way of example, vaccinia virus recombinants expressing thegenes can be constructed as follows. The DNA encoding the particularsynthetic HIV polypeptide coding sequence is first inserted into anappropriate vector so that it is adjacent to a vaccinia promoter andflanking vaccinia DNA sequences, such as the sequence encoding thymidinekinase (TK). This vector is then used to transfect cells which aresimultaneously infected with vaccinia. Homologous recombination servesto insert the vaccinia promoter plus the gene encoding the codingsequences of interest into the viral genome. The resultingTK-recombinant can be selected by culturing the cells in the presence of5-bromodeoxyuridine and picking viral plaques resistant thereto.

Alternatively, avipoxviruses, such as the fowlpox and canarypox viruses,can also be used to deliver the genes. Recombinant avipox viruses,expressing immunogens from mammalian pathogens, are known to conferprotective immunity when administered to non-avian species. The use ofan avipox vector is particularly desirable in human and other mammalianspecies since members of the avipox genus can only productivelyreplicate in susceptible avian species and therefore are not infectivein mammalian cells. Methods for producing recombinant avipoxviruses areknown in the art and employ genetic recombination, as described abovewith respect to the production of vaccinia viruses. See, e.g., WO91/12882; WO 89/03429; and WO 92/03545.

Molecular conjugate vectors, such as the adenovirus chimeric vectorsdescribed in Michael et al., J. Biol. Chem. (1993) 268:6866-6869 andWagner et al., Proc. Natl. Acad. Sci. USA (1992) 89:6099-6103, can alsobe used for gene delivery.

Members of the Alphavirus genus, such as, but not limited to, vectorsderived from the Sindbis, Semliki Forest, and Venezuelan EquineEncephalitis viruses, will also find use as viral vectors for deliveringthe polynucleotides of the present invention (for example, a syntheticGag-polypeptide encoding expression cassette). For a description ofSindbis-virus derived vectors useful for the practice of the instantmethods, see, Dubensky et al., J. Virol. (1996) 70:508-519; andInternational Publication Nos. WO 95/07995 and WO 96/17072; as well as,Dubensky, Jr., T. W., et al., U.S. Pat. No. 5,843,723, issued Dec. 1,1998, and Dubensky, Jr., T. W., U.S. Pat. No. 5,789,245, issued Aug. 4,1998, both herein incorporated by reference. Preferred expressionsystems include, but are not limited to, eucaryotic layered vectorinitiation systems (e.g., U.S. Pat. No. 6,015,686, U.S. Pat. No.5,814,482, U.S. Pat. No. 6,015,694, U.S. Pat. No. 5,789,245, EP1029068A2, WO 9918226A2/A3, EP 00907746A2, WO 9738087A2, all hereinincorporated by reference in their entireties).

A vaccinia based infection/transfection system can be conveniently usedto provide for inducible, transient expression of the coding sequencesof interest in a host cell. In this system, cells are first infected invitro with a vaccinia virus recombinant that encodes the bacteriophageT7 RNA polymerase. This polymerase displays exquisite specificity inthat it only transcribes templates bearing T7 promoters. Followinginfection, cells are transfected with the polynucleotide of interest,driven by a T7 promoter. The polymerase expressed in the cytoplasm fromthe vaccinia virus recombinant transcribes the transfected DNA into RNAwhich is then translated into protein by the host translationalmachinery. The method provides for high level, transient, cytoplasmicproduction of large quantities of RNA and its translation products. See,e.g., Elroy-Stein and Moss, Proc. Natl. Acad. Sci. USA (1990)87:6743-6747; Fuerst et al., Proc. Natl. Acad. Sci. USA (1986)83:8122-8126.

As an alternative approach to infection with vaccinia or avipox virusrecombinants, or to the delivery of genes using other viral vectors, anamplification system can be used that will lead to high level expressionfollowing introduction into host cells. Specifically, a T7 RNApolymerase promoter preceding the coding region for T7 RNA polymerasecan be engineered. Translation of RNA derived from this template willgenerate T7 RNA polymerase which in turn will transcribe more template.Concomitantly, there will be a cDNA whose expression is under thecontrol of the T7 promoter. Thus, some of the T7 RNA polymerasegenerated from translation of the amplification template RNA will leadto transcription of the desired gene. Because some T7 RNA polymerase isrequired to initiate the amplification, T7 RNA polymerase can beintroduced into cells along with the template(s) to prime thetranscription reaction. The polymerase can be introduced as a protein oron a plasmid encoding the RNA polymerase. For a further discussion of T7systems and their use for transforming cells, see, e.g., InternationalPublication No. WO 94/26911; Studier and Moffatt, J. Mol. Biol. (1986)189:113-130; Deng and Wolff, Gene (1994) 143:245-249; Gao et al.,Biochem. Biophys. Res. Commun. (1994) 200:1201-1206; Gao and Huang, Nuc.Acids Res. (1993) 21:2867-2872; Chen et al., Nuc. Acids Res. (1994)22:2114-2120; and U.S. Pat. No. 5,135,855.

Delivery of the expression cassettes of the present invention can alsobe accomplished using eucaryotic expression vectors comprisingCMV-derived elements, such vectors include, but are not limited to, thefollowing: pCMVKm2, pCMV-link pCMVPLEdhfr, and pCMV6a (all describedabove).

Synthetic expression cassettes of interest can also be delivered withouta viral vector. For example, the synthetic expression cassette can bepackaged in liposomes prior to delivery to the subject or to cellsderived therefrom. Lipid encapsulation is generally accomplished usingliposomes which are able to stably bind or entrap and retain nucleicacid. The ratio of condensed DNA to lipid preparation can vary but willgenerally be around 1:1 (mg DNA:micromoles lipid), or more of lipid. Fora review of the use of liposomes as carriers for delivery of nucleicacids, see, Hug and Sleight, Biochim. Biophys. Acta. (1991) 1097:1-17;Straubinger et al., in Methods of Enzymology (1983), Vol. 101, pp.512-527.

Liposomal preparations for use in the present invention include cationic(positively charged), anionic (negatively charged) and neutralpreparations, with cationic liposomes particularly preferred. Cationicliposomes have been shown to mediate intracellular delivery of plasmidDNA (Felgner et al., Proc. Natl. Acad. Sci. USA (1987) 84:7413-7416);mRNA (Malone et al., Proc. Natl. Acad. Sci. USA (1989) 86:6077-6081);and purified transcription factors (Debs et al., J. Biol. Chem. (1990)265:10189-10192), in functional form.

Cationic liposomes are readily available. For example,N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes areavailable under the trademark Lipofectin, from GIBCO BRL, Grand Island,N.Y. (See, also, Felgner et al., Proc. Natl. Acad. Sci. USA (1987)84:7413-7416). Other commercially available lipids include (DDAB/DOPE)and DOTAP/DOPE (Boerhinger). Other cationic liposomes can be preparedfrom readily available materials using techniques well known in the art.See, e.g., Szoka et al., Proc. Natl. Acad. Sci. USA (1978) 75:4194-4198;PCT Publication No. WO 90/11092 for a description of the synthesis ofDOTAP (1,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes.

Similarly, anionic and neutral liposomes are readily available, such as,from Avanti Polar Lipids (Birmingham, Ala.), or can be easily preparedusing readily available materials. Such materials include phosphatidylcholine, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidyl glycerol (DOPG),dioleoylphoshatidyl ethanolamine (DOPE), among others. These materialscan also be mixed with the DOTMA and DOTAP starting materials inappropriate ratios. Methods for making liposomes using these materialsare well known in the art.

The liposomes can comprise multilammelar vesicles (MLVs), smallunilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs). Thevarious liposome-nucleic acid complexes are prepared using methods knownin the art. See, e.g., Straubinger et al., in METHODS OF IMMUNOLOGY(1983), Vol. 101, pp. 512-527; Szoka et al., Proc. Natl. Acad. Sci. USA(1978) 75:4194-4198; Papahadjopoulos et al., Biochim. Biophys. Acta(1975) 394:483; Wilson et al., Cell (1979) 17:77); Deamer and Bangham,Biochim. Biophys. Acta (1976) 443:629; Ostro et al., Biochem. Biophys.Res. Commun. (1977) 76:836; Fraley et al., Proc. Natl. Acad. Sci. USA(1979) 76:3348); Enoch and Strittmatter, Proc. Natl. Acad. Sci. USA(1979) 76:145); Fraley et al., J. Biol. Chem. (1980) 255:10431; Szokaand Papahadjopoulos, Proc. Natl. Acad. Sci. USA (1978) 75:145; andSchaefer-Ridder et al., Science (1982) 215:166.

The DNA and/or protein antigen(s) can also be delivered in cochleatelipid compositions similar to those described by Papahadjopoulos et al.,Biochem. Biophys. Acta. (1975) 394:483-491. See, also, U.S. Pat. Nos.4,663,161 and 4,871,488.

The synthetic expression cassette of interest may also be encapsulated,adsorbed to, or associated with, particulate carriers. Such carrierspresent multiple copies of a selected antigen to the immune system andpromote trapping and retention of antigens in local lymph nodes. Theparticles can be phagocytosed by macrophages and can enhance antigenpresentation through cytokine release. Examples of particulate carriersinclude those derived from polymethyl methacrylate polymers, as well asmicroparticles derived from poly(lactides) andpoly(lactide-co-glycolides), known as PLG. See, e.g., Jeffery et al.,Pharm. Res. (1993) 10:362-368; McGee J P, et al., J Microencapsul.14(2): 197-210, 1997; O'Hagan D T, et al., Vaccine 11(2): 149-54, 1993.Suitable microparticles may also be manufactured in the presence ofcharged detergents, such as anionic or cationic detergents, to yieldmicroparticles with a surface having a net negative or a net positivecharge. For example, microparticles manufactured with anionicdetergents, such as hexadecyltrimethylammonium bromide (CTAB), i.e.CTAB-PLG microparticles, adsorb negatively charged macromolecules, suchas DNA. (see, e.g., Int'l Application Number PCT/US99/17308).

Furthermore, other particulate systems and polymers can be used for thein vivo or ex vivo delivery of the gene of interest. For example,polymers such as polylysine, polyarginine, polyornithine, spermine,spermidine, as well as conjugates of these molecules, are useful fortransferring a nucleic acid of interest. Similarly, DEAEdextran-mediated transfection, calcium phosphate precipitation orprecipitation using other insoluble inorganic salts, such as strontiumphosphate, aluminum silicates including bentonite and kaolin, chromicoxide, magnesium silicate, talc, and the like, will find use with thepresent methods. See, e.g., Feigner, P. L., Advanced Drug DeliveryReviews (1990) 5:163-187, for a review of delivery systems useful forgene transfer. Peptoids (Zuckerman, R. N., et al., U.S. Pat. No.5,831,005, issued Nov. 3, 1998, herein incorporated by reference) mayalso be used for delivery of a construct of the present invention.

Additionally, biolistic delivery systems employing particulate carrierssuch as gold and tungsten, are especially useful for deliveringsynthetic expression cassettes of the present invention. The particlesare coated with the synthetic expression cassette(s) to be delivered andaccelerated to high velocity, generally under a reduced atmosphere,using a gun powder discharge from a “gene gun.” For a description ofsuch techniques, and apparatuses useful therefore, see, e.g., U.S. Pat.Nos. 4,945,050; 5,036,006; 5,100,792; 5,179,022; 5,371,015; and5,478,744. Also, needle-less injection systems can be used (Davis, H.L., et al, Vaccine 12:1503-1509, 1994; Bioject, Inc., Portland, Oreg.).

Recombinant vectors carrying a synthetic expression cassette of thepresent invention are formulated into compositions for delivery to thevertebrate subject. These compositions may either be prophylactic (toprevent infection) or therapeutic (to treat disease after infection).The compositions will comprise a “therapeutically effective amount” ofthe gene of interest such that an amount of the antigen can be producedin vivo so that an immune response is generated in the individual towhich it is administered. The exact amount necessary will vary dependingon the subject being treated; the age and general condition of thesubject to be treated; the capacity of the subject's immune system tosynthesize antibodies; the degree of protection desired; the severity ofthe condition being treated; the particular antigen selected and itsmode of administration, among other factors. An appropriate effectiveamount can be readily determined by one of skill in the art. Thus, a“therapeutically effective amount” will fall in a relatively broad rangethat can be determined through routine trials.

The compositions will generally include one or more “pharmaceuticallyacceptable excipients or vehicles” such as water, saline, glycerol,polyethyleneglycol, hyaluronic acid, ethanol, etc. Additionally,auxiliary substances, such as wetting or emulsifying agents, pHbuffering substances, and the like, may be present in such vehicles.Certain facilitators of nucleic acid uptake and/or expression can alsobe included in the compositions or coadministered, such as, but notlimited to, bupivacaine, cardiotoxin and sucrose.

Once formulated, the compositions of the invention can be administereddirectly to the subject (e.g., as described above) or, alternatively,delivered ex vivo, to cells derived from the subject, using methods suchas those described above. For example, methods for the ex vivo deliveryand reimplantation of transformed cells into a subject are known in theart and can include, e.g., dextran-mediated transfection, calciumphosphate precipitation, polybrene mediated transfection, lipofectamineand LT-1 mediated transfection, protoplast fusion, electroporation,encapsulation of the polynucleotide(s) (with or without thecorresponding antigen) in liposomes, and direct microinjection of theDNA into nuclei.

Direct delivery of synthetic expression cassette compositions in vivowill generally be accomplished with or without viral vectors, asdescribed above, by injection using either a conventional syringe or agene gun, such as the Accell® gene delivery system (PowderJectTechnologies, Inc., Oxford, England). The constructs can be injectedeither subcutaneously, epidermally, intradermally, intramucosally suchas nasally, rectally and vaginally, intraperitoneally, intravenously,orally or intramuscularly. Delivery of DNA into cells of the epidermisis particularly preferred as this mode of administration provides accessto skin-associated lymphoid cells and provides for a transient presenceof DNA in the recipient. Other modes of administration include oral andpulmonary administration, suppositories, needle-less injection,transcutaneous and transdermal applications. Dosage treatment may be asingle dose schedule or a multiple dose schedule. Administration ofnucleic acids may also be combined with administration of peptides orother substances.

Exemplary immunogenicity studies are presented in Examples 4, 5, 6, 9,10, 11, and 12.

2.4.2 Ex Vivo Delivery of the Synthetic Expression Cassettes of thePresent Invention

In one embodiment, T cells, and related cell types (including but notlimited to antigen presenting cells, such as, macrophage, monocytes,lymphoid cells, dendritic cells, B-cells, T-cells, stem cells, andprogenitor cells thereof), can be used for ex vivo delivery of thesynthetic expression cassettes of the present invention. T cells can beisolated from peripheral blood lymphocytes (PBLs) by a variety ofprocedures known to those skilled in the art. For example, T cellpopulations can be “enriched” from a population of PBLs through theremoval of accessory and B cells. In particular, T cell enrichment canbe accomplished by the elimination of non-T cells using anti-MHC classII monoclonal antibodies. Similarly, other antibodies can be used todeplete specific populations of non-T cells. For example, anti-Igantibody molecules can be used to deplete B cells and anti-MacI antibodymolecules can be used to deplete macrophages.

T cells can be further fractionated into a number of differentsubpopulations by techniques known to those skilled in the art. Twomajor subpopulations can be isolated based on their differentialexpression of the cell surface markers CD4 and CD8. For example,following the enrichment of T cells as described above, CD4⁺ cells canbe enriched using antibodies specific for CD4 (see Coligan et al.,supra). The antibodies may be coupled to a solid support such asmagnetic beads. Conversely, CD8+ cells can be enriched through the useof antibodies specific for CD4 (to remove CD4⁺ cells), or can beisolated by the use of CD8 antibodies coupled to a solid support. CD4lymphocytes from HIV-1 infected patients can be expanded ex vivo, beforeor after transduction as described by Wilson et. al. (1995) J. Infect.Dis. 172:88.

Following purification of T cells, a variety of methods of geneticmodification known to those skilled in the art can be performed usingnon-viral or viral-based gene transfer vectors constructed as describedherein. For example, one such approach involves transduction of thepurified T cell population with vector-containing supernatant ofcultures derived from vector producing cells. A second approach involvesco-cultivation of an irradiated monolayer of vector-producing cells withthe purified T cells. A third approach involves a similar co-cultivationapproach; however, the purified T cells are pre-stimulated with variouscytokines and cultured 48 hours prior to the co-cultivation with theirradiated vector producing cells. Pre-stimulation prior to suchtransduction increases effective gene transfer (Nolta et al. (1992) Exp.Hematol. 20:1065). Stimulation of these cultures to proliferate alsoprovides increased cell populations for re-infusion into the patient.Subsequent to co-cultivation, T cells are collected from the vectorproducing cell monolayer, expanded, and frozen in liquid nitrogen.

Gene transfer vectors, containing one or more synthetic expressioncassette of the present invention (associated with appropriate controlelements for delivery to the isolated T cells) can be assembled usingknown methods and following the guidance of the present specification.

Selectable markers can also be used in the construction of gene transfervectors. For example, a marker can be used which imparts to a mammaliancell transduced with the gene transfer vector resistance to a cytotoxicagent. The cytotoxic agent can be, but is not limited to, neomycin,aminoglycoside, tetracycline, chloramphenicol, sulfonamide, actinomycin,netropsin, distamycin A, anthracycline, or pyrazinamide. For example,neomycin phosphotransferase II imparts resistance to the neomycinanalogue geneticin (G418).

The T cells can also be maintained in a medium containing at least onetype of growth factor prior to being selected. A variety of growthfactors are known in the art which sustain the growth of a particularcell type. Examples of such growth factors are cytokine mitogens such asrIL-2, IL-10, IL-12, and IL-15, which promote growth and activation oflymphocytes. Certain types of cells are stimulated by other growthfactors such as hormones, including human chorionic gonadotropin (hCG)and human growth hormone. The selection of an appropriate growth factorfor a particular cell population is readily accomplished by one of skillin the art.

For example, white blood cells such as differentiated progenitor andstem cells are stimulated by a variety of growth factors. Moreparticularly, IL-3, IL-4, IL-5, IL-6, IL-9, GM-CSF, M-CSF, and G-CSF,produced by activated T_(H) and activated macrophages, stimulate myeloidstem cells, which then differentiate into pluripotent stem cells,granulocyte-monocyte progenitors, eosinophil progenitors, basophilprogenitors, megakaryocytes, and erythroid progenitors. Differentiationis modulated by growth factors such as GM-CSF, IL-3, IL-6, IL-1, andEPO.

Pluripotent stem cells then differentiate into lymphoid stem cells, bonemarrow stromal cells, T cell progenitors, B cell progenitors,thymocytes, T_(H) Cells, T_(C) cells, and B cells. This differentiationis modulated by growth factors such as IL-3, IL-4, IL-6, IL-7, GM-CSF,M-CSF, G-CSF, IL-2, and IL-5.

Granulocyte-monocyte progenitors differentiate to monocytes,macrophages, and neutrophils. Such differentiation is modulated by thegrowth factors GM-CSF, M-CSF, and IL-8. Eosinophil progenitorsdifferentiate into eosinophils. This process is modulated by GM-CSF andIL-5.

The differentiation of basophil progenitors into mast cells andbasophils is modulated by GM-CSF, IL-4, and IL-9. Megakaryocytes produceplatelets in response to GM-CSF, EPO, and IL-6. Erythroid progenitorcells differentiate into red blood cells in response to EPO.

Thus, during activation by the CD3-binding agent, T cells can also becontacted with a mitogen, for example a cytokine such as IL-2. Inparticularly preferred embodiments, the IL-2 is added to the populationof T cells at a concentration of about 50 to 100 μg/ml. Activation withthe CD3-binding agent can be carried out for 2 to 4 days.

Once suitably activated, the T cells are genetically modified bycontacting the same with a suitable gene transfer vector underconditions that allow for transfection of the vectors into the T cells.Genetic modification is carried out when the cell density of the T cellpopulation is between about 0.1×10⁶ and 5×10⁶, preferably between about0.5×10⁶ and 2×10⁶. A number of suitable viral and nonviral-based genetransfer vectors have been described for use herein.

After transduction, transduced cells are selected away fromnon-transduced cells using known techniques. For example, if the genetransfer vector used in the transduction includes a selectable markerwhich confers resistance to a cytotoxic agent, the cells can becontacted with the appropriate cytotoxic agent, whereby non-transducedcells can be negatively selected away from the transduced cells. If theselectable marker is a cell surface marker, the cells can be contactedwith a binding agent specific for the particular cell surface marker,whereby the transduced cells can be positively selected away from thepopulation. The selection step can also entail fluorescence-activatedcell sorting (FACS) techniques, such as where FACS is used to selectcells from the population containing a particular surface marker, or theselection step can entail the use of magnetically responsive particlesas retrievable supports for target cell capture and/or backgroundremoval.

More particularly, positive selection of the transduced cells can beperformed using a FACS cell sorter (e.g. a FACSVantage™ Cell Sorter,Becton Dickinson Immunocytometry Systems, San Jose, Calif.) to sort andcollect transduced cells expressing a selectable cell surface marker.Following transduction, the cells are stained with fluorescent-labeledantibody molecules directed against the particular cell surface marker.The amount of bound antibody on each cell can be measured by passingdroplets containing the cells through the cell sorter. By imparting anelectromagnetic charge to droplets containing the stained cells, thetransduced cells can be separated from other cells. The positivelyselected cells are then harvested in sterile collection vessels. Thesecell sorting procedures are described in detail, for example, in theFACSVantage™ Training Manual, with particular reference to sections 3-11to 3-28 and 10-1 to 10-17.

Positive selection of the transduced cells can also be performed usingmagnetic separation of cells based on expression or a particular cellsurface marker. In such separation techniques, cells to be positivelyselected are first contacted with specific binding agent (e.g., anantibody or reagent the interacts specifically with the cell surfacemarker). The cells are then contacted with retrievable particles (e.g.,magnetically responsive particles) which are coupled with a reagent thatbinds the specific binding agent (that has bound to the positive cells).The cell-binding-agent-particle complex can then be physically separatedfrom non-labeled cells, for example using a magnetic field. When usingmagnetically responsive particles, the labeled cells can be retained ina container using a magnetic filed while the negative cells are removed.These and similar separation procedures are known to those of ordinaryskill in the art.

Expression of the vector in the selected transduced cells can beassessed by a number of assays known to those skilled in the art. Forexample, Western blot or Northern analysis can be employed depending onthe nature of the inserted nucleotide sequence of interest. Onceexpression has been established and the transformed T cells have beentested for the presence of the selected synthetic expression cassette,they are ready for infusion into a patient via the peripheral bloodstream.

The invention includes a kit for genetic modification of an ex vivopopulation of primary mammalian cells. The kit typically contains a genetransfer vector coding for at least one selectable marker and at leastone synthetic expression cassette contained in one or more containers,ancillary reagents or hardware, and instructions for use of the kit.

2.4.3 Further Delivery Regimes

Any of the polynucleotides (e.g., expression cassettes) or polypeptidesdescribed herein (delivered by any of the methods described above) canalso be used in combination with other DNA delivery systems and/orprotein delivery systems. Non-limiting examples includeco-administration of these molecules, for example, in prime-boostmethods where one or more molecules are delivered in a “priming” stepand, subsequently, one or more molecules are delivered in a “boosting”step. In certain embodiments, the delivery of one or more nucleicacid-containing compositions and is followed by delivery of one or morenucleic acid-containing compositions and/or one or morepolypeptide-containing compositions (e.g., polypeptides comprising HIVantigens). In other embodiments, multiple nucleic acid “primes” (of thesame or different nucleic acid molecules) can be followed by multiplepolypeptide “boosts” (of the same or different polypeptides). Otherexamples include multiple nucleic acid administrations and multiplepolypeptide administrations.

In any method involving co-administration, the various compositions canbe delivered in any order. Thus, in embodiments including delivery ofmultiple different compositions or molecules, the nucleic acids need notbe all delivered before the polypeptides. For example, the priming stepmay include delivery of one or more polypeptides and the boostingcomprises delivery of one or more nucleic acids and/or one morepolypeptides. Multiple polypeptide administrations can be followed bymultiple nucleic acid administrations or polypeptide and nucleic acidadministrations can be performed in any order. In any of the embodimentsdescribed herein, the nucleic acid molecules can encode all, some ornone of the polypeptides. Thus, one or more or the nucleic acidmolecules (e.g., expression cassettes) described herein and/or one ormore of the polypeptides described herein can be co-administered in anyorder and via any administration routes. Therefore, any combination ofpolynucleotides and/or polypeptides described herein can be used togenerate elicit an immune reaction.

3.0 Improved HIV-1 Gag and Pol Expression Cassettes

While not desiring to be bound by any particular model, theory, orhypothesis, the following information is presented to provide a morecomplete understanding of the present invention.

The world health organization (WHO) estimated the number of peopleworldwide that are infected with HIV-1 to exceed 36.1 million. Thedevelopment of a safe and effective HIV vaccine is therefore essentialat this time. Recent studies have demonstrated the importance of CTL incontrolling the HIV-1 replication in infected patients. Furthermore, CTLreactivity with multiple HIV antigens will be necessary for theeffective control of virus replication. Experiments performed in supportof the present invention suggest that the inclusion of HIV-1 Gag andPol, beside Env for the induction of neutralizing antibodies, into thevaccine is useful.

To increase the potency of HIV-1 vaccine candidates, codon modified Gagand Pol expression cassettes were designed, either for Gag alone or Gagplus Pol. To evaluate possible differences in expression and potency,the expression of these constructs was analyzed and immunogenicitystudies carried out in mice.

Several expression cassettes encoding Gag and Pol were designed,including, but not limited to, the following: GagProtease,GagPolΔintegrase with frameshift (gagFSpol), and GagPolΔintegrasein-frame (gagpol). Versions of GagPolΔintegrase in-frame were alsodesigned with attenuated (Att) or non-functional Protease (Ina). Thenucleic acid sequences were codon modified to correspond to the codonusage of highly expressed human genes. Mice were immunized with titratedDNA doses and humoral and cellular immune responses evaluated by ELISAand intracellular cytokine staining (Example 10).

The immune responses in mice has been seen to be correlated withrelative levels of expression in vitro. Vaccine studies in rhesusmonkeys will further address immune responses and expression levels invivo.

4.0 Enhanced Vaccine Technologies for the Induction of PotentNeutralizing Antibodies and Cellular Immune Responses Against HIV.

While not desiring to be bound by any particular model, theory, orhypothesis, the following information is presented to provide a morecomplete understanding of the present invention.

Protection against HIV infection will likely require potent and broadlyreactive pre-existing neutralizing antibodies in vaccinated individualsexposed to a virus challenge. Although cellular immune responses aredesirable to control viremia in those who get infected, protectionagainst infection has not been demonstrated for vaccine approaches thatrely exclusively on the induction of these responses. For this reason,experiments performed in support of the present invention useprime-boost approaches that employ novel V-deleted envelope antigensfrom primary HIV isolates (e.g., R5 subtype B (HIV-1_(SF162)) andsubtype C(HIV-1_(TVI)) strains). These antigens were delivered byenhanced DNA [polyactide co-glycolide (PLG) microparticle formulationsor electroporation] or alphavirus replicon particle-based vaccineapproaches, followed by booster immunizations with Env proteins in MF59adjuvant. Efficient in vivo expression of plasmid encoded genes byelectrical permeabilization has been described (see, e.g., Zucchelli etal. (2000) J. Virol. 74:11598-11607; Banga et al. (1998) TrendsBiotechnol. 10:408-412; Heller et al. (1996) Febs Lett. 389:225-228;Mathiesen et al. (1999) Gene Ther. 4:508-514; Mir et al. (1999) Proc.Nat'l Acad. Sci. USA 8:4262-4267; Nishi et al. (1996) Cancer Res.5:1050-1055). Both native and V-deleted monomeric (gp120) and oligomeric(o-gp140) forms of protein from the SF162 strain were tested asboosters. All protein preparations were highly purified and extensivelycharacterized by biophysical and immunochemical methodologies. Resultsfrom rabbit and primate immunogenicity studies indicated that, whereasneutralizing antibody responses could be consistently induced againstthe parental non-V2-deleted SF162 virus, the induction of responsesagainst heterologous HIV strains improved with deletion of the V2 loopof the immunogens. Moreover, using these prime-boost vaccine regimens,potent HIV antigen-specific CD4+ and CD8+ T-cell responses were alsodemonstrated.

Based on these findings, V2-deleted envelope DNA and protein vaccineswere chosen for advancement toward clinical evaluation. Similarapproaches for immunization may be employed using, for example, nucleicacid immunization employing the synthetic HIV polynucleotides of thepresent invention coupled with corresponding or heterologous HIV-derivedpolypeptide boosts.

One embodiment of this aspect of the present invention may be describedgenerally as follows. Antigens are selected for the vaccinecomposition(s). Env polypeptides are typically employed in a firstantigenic composition used to induce an immune response. Further, Gagpolypeptides are typically employed in a second antigenic compositionused to induce an immune response. The second antigenic composition mayinclude further HIV-derived polypeptide sequences, including, but notlimited to, Pol, Tat, Rev, Nef, Vif, Vpr, and/or Vpu sequences. A DNAprime vaccination is typically performed with the first and secondantigenic compositions. Further DNA vaccinations with one or more of theantigenic compositions may also be included at selected time intervals.The prime is typically followed by at least one boost. The boost may,for example, include adjuvanted HIV-derived polypeptides (e.g.,corresponding to those used for the DNA vaccinations), coding sequencesfor HIV-derived polypeptides (e.g., corresponding to those used for theDNA vaccinations) encoded by a viral vector, further DNA vaccinations,and/or combinations of the foregoing. In one embodiment, a DNA prime isadministered with a first antigenic composition (e.g., a DNA constructencoding an Envelope polypeptide) and second antigenic composition(e.g., a DNA construct encoding a Gag polypeptide, a Pol polypeptide, aTat polypeptide, a Nef polypeptide, and a Rev polypeptide). The DNAconstruct for use in the prime may, for example, comprise a CMV promoteroperably linked to the polynucleotide encoding the polypeptide sequence.The DNA prime is followed by a boost, for example, an adjuvantedEnvelope polypeptide boost and a viral vector boost (where the viralvector encodes, e.g., a Gag polypeptide, a Pol polypeptide, a Tatpolypeptide, a Nef polypeptide, and a Rev polypeptide). Alternately (orin addition), the boost may be an adjuvanted Gag polypeptide, Polpolypeptide, Tat polypeptide, Nef polypeptide, and Rev polypeptide boostand a viral vector boost (where the viral vector encodes, e.g., anEnvelope polypeptide). The boost may include all polypeptide antigenswhich were encoded in the DNA prime; however, this is not required.Further, different polypeptide antigens may be used in the boostrelative to the initial vaccination and visa versa. Further, the initialvaccination may be a viral vector rather than a DNA construct.

Some factors that may be considered in HIV envelope vaccine design areas follows. Envelope-based vaccines have demonstrated protection againstinfection in non-human primate models. Passive antibody studies havedemonstrated protection against HIV infection in the presence ofneutralizing antibodies against the virus challenge stock. Vaccines thatexclude Env generally confer less protective efficacy. Experimentsperformed in support of the present invention have demonstrated thatmonomeric gp120 protein-derived from the SF2 lab strain providedneutralization of HIV-1 lab strains and protection against viruschallenges in primate models. Primary gp120 protein derived from That Efield strains provided cross-subtype neutralization of lab strains.Primary sub-type B oligomeric o-gp140 protein provided partialneutralization of subtype B primary (field) isolates. Primary sub-type Bo-gp140ΔV2 DNA prime plus protein boost provided potent neutralizationof diverse subtype B primary isolates and protection against viruschallenge in primate models. Primary sub-type C o-gp140 and o-gp140ΔV2likely provide similar results to those just described for sub-type B.

Vaccine strategies for induction of potent, broadly reactive,neutralizing antibodies may be assisted by construction of Envelopepolypeptide structures that expose conserved neutralizing epitopes, forexample, variable-region deletions and de-glycosylations, envelopeprotein-receptor complexes, rational design based on crystal structure(e.g., β-sheet deletions), and gp41-fusion domain based immunogens.

Stable CHO cell lines for envelope protein production have beendeveloped using optimized envelope polypeptide coding sequences,including, but not limited to, the following: gp120, o-gp140, gp120ΔV2,o-gp140ΔV2, gp120ΔV1V2, o-gp140ΔVIV2.

In addition, following prime-boost regimes (such as those describedabove) appear to be beneficial to help reduce viral load in infectedsubjects, as well as possibly slow or prevent progression of HIV-relateddisease (relative to untreated subjects).

Exemplary antigenic compositions and immunogenicity studies arepresented in Examples 9, 10, 11, and 12.

EXPERIMENTAL

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should, of course, be allowed for.

Example 1 Generation of Synthetic Expression Cassettes

A. Generating Synthetic Polynucleotides

The polynucleotide sequences of the present invention were manipulatedto maximize expression of their gene products. The order of thefollowing steps may vary.

First, the HIV-1 codon usage pattern was modified so that the resultingnucleic acid coding sequence was comparable to codon usage found inhighly expressed human genes. The HIV codon usage reflects a highcontent of the nucleotides A or T of the codon-triplet. The effect ofthe HIV-1 codon usage is a high AT content in the DNA sequence thatresults in a high AU content in the RNA and in a decreased translationability and instability of the mRNA. In comparison, highly expressedhuman codons prefer the nucleotides G or C. The wild-type sequences weremodified to be comparable to codon usage found in highly expressed humangenes.

Second, for some genes non-functional variants were created. In thefollowing table (Table B) mutations affecting the activity of severalHIV genes are disclosed. All references cited are herein incorporated byreference. TABLE B Gene “Region” Exemplary Mutations Pol prot Att =Reduced activity by attenuation of Protease (Thr26Ser) (e.g., Konvalinkaet al., 1995, J Virol 69: 7180-86) Ina = Mutated Protease, nonfunctionalenzyme (Asp25Ala)(e.g., Konvalinka et al., 1995, J Virol 69: 7180-86) RTYM = Deletion of catalytic center (YMDD_AP; SEQ ID NO: 7) (e.g.,Biochemistry, 1995, 34, 5351, Patel et. al.) WM = Deletion of primergrip region (WMGY_PI; SEQ ID NO: 8)) (e.g., J Biol Chem, 272, 17, 11157,Palaniappan, et. al., 1997) RNase no direct mutations, RnaseH isaffected by “WM” mutation in RT Integrase 1) Mutation of HHCC domain,Cys40Ala (e.g., Wiskerchen et. al., 1995, J Virol, 69: 376). 2.)Inactivation catalytic center, Asp64Ala, Asp116Ala, Glu152Ala (e.g.,Wiskerchen et. al., 1995, J Virol, 69: 376). 3) Inactivation of minimalDNA binding domain (MDBD), deletion of Trp235(e.g., Ishikawa et. al.,1999, J Virol, 73: 4475). Constructs int.opt.mut.SF2 and int.opt.mut_C(South Africa TV1) both contain all these mutations (1, 2, and 3) EnvMutations in cleavage site (e.g., mut1-4, 7) Mutations in glycosylationsite (e.g., GM mutants, for example, change Q residue in V1 and/or V2 toN residue; may also be designated by residue altered in sequence) TatMutants of Tat in transactivation domain (e.g., Caputo et al., 1996,Gene Ther. 3: 235) cys22 mutant (Cys22Gly) = TatC22 cys37 mutant(Cys37Ser) = TatC37 cys22/37 double mutant = TatC22/37 Rev Mutations inRev domains (e.g., Thomas et al., 1998, J Virol. 72: 2935-44) Mutationin RNA binding-nuclear localization ArgArg38,39AspLeu = M5 Mutation inactivation domain LeuGlu78,79AspLeu = M10 Nef Mutations ofmyristoyilation signal and in oligomerization domain: 1. Single pointmutation myristoyilation signal: Gly-to-Ala = -Myr 2. Deletion ofN-terminal first 18 (sub-type B, e.g., SF162) or 19 (sub-type C, e.g.,South Africa clones) amino acids: -Myr18 or -Myr19 (respectively) (e.g.,Peng and Robert-Guroff, 2001, Immunol Letters 78: 195-200) Single pointmutation oligomerization: (e.g., Liu et al., 2000, J Virol 74: 5310-19)Asp125Gly (sub B SF162) or Asp124Gly (sub C South Africa clones)Mutations affecting (1) infectivity (replication) of HIV- virions and/or(2) CD4 down regulation. (e.g., Lundquist et al. (2002) J Virol. 76(9):4625-33) Vif Mutations of Vif: e.g., Simon et al., 1999, J Virol 73:2675-81 Vpr Mutations of Vpr: e.g., Singh et al., 2000, J Virol 74:10650-57 Vpu Mutations of Vpu: e.g., Tiganos et al., 1998, Virology 251:96-107

Constructs comprising some of these mutations are described herein. Vif,vpr and vpu synthetic constructs are described. Reducing or eliminatingthe function of the associated gene products can be accomplishedemploying the teachings set forth in the above table, in view of theteachings of the present specification.

In one embodiment of the invention, the full length coding region of theGag-polymerase sequence is included with the synthetic Gag sequences inorder to increase the number of epitopes for virus-like particlesexpressed by the synthetic, optimized Gag expression cassette. Becausesynthetic HIV-1 Gag-polymerase expresses the potentially deleteriousfunctional enzymes reverse transcriptase (RT) and integrase (INT) (inaddition to the structural proteins and protease), it is important toinactivate RT and INT functions. Several in-frame deletions in the RTand INT reading frame can be made to achieve catalytic nonfunctionalenzymes with respect to their RT and INT activity. {Jay. A. Levy(Editor) (1995) The Retroviridae, Plenum Press, New York. ISBN0-30645033X. Pages 215-20; Grimison, B. and Laurence, J. (1995), JournalOf Acquired Immune Deficiency Syndromes and Human Retrovirology9(1):58-68; Wakefield, J. K., et al., (1992) Journal Of Virology66(11):6806-6812; Esnouf, R., et al., (1995) Nature Structural Biology2(4):303-308; Maignan, S., et al., (1998) Journal Of Molecular Biology282(2):359-368; Katz, R. A. and Skalka, A. M. (1994) Annual Review OfBiochemistry 73 (1994); Jacobo-Molina, A., et al., (1993) Proceedings Ofthe National Academy Of Sciences Of the United States Of America90(13):6320-6324; Hickman, A. B., et al., (1994) Journal Of BiologicalChemistry 269(46):29279-29287; Goldgur, Y., et al., (1998) ProceedingsOf the National Academy Of Sciences Of the United States Of America95(16):9150-9154; Goette, M., et al., (1998) Journal Of BiologicalChemistry 273(17):10139-10146; Gorton, J. L., et al., (1998) Journal ofVirology 72(6):5046-5055; Engelman, A., et al., (1997) Journal OfVirology 71(5):3507-3514; Dyda, F., et al., Science 266(5193):1981-1986;Davies, J. F., et al., (1991) Science 252(5002):88-95; Bujacz, G., etal., (1996) Febs Letters 398(2-3):175-178; Beard, W. A., et al., (1996)Journal Of Biological Chemistry 271(21):12213-12220; Kohlstaedt, L. A.,et al., (1992) Science 256(5065):1783-1790; Krug, M. S, and Berger, S.L. (1991) Biochemistry 30(44): 10614-10623; Mazumder, A., et al., (1996)Molecular Pharmacology 49(4):621-628; Palaniappan, C., et al., (1997)Journal Of Biological Chemistry 272(17): 11157-11164; Rodgers, D. W., etal., (1995) Proceedings Of the National Academy Of Sciences Of theUnited States Of America 92(4): 1222-1226; Sheng, N. and Dennis, D.(1993) Biochemistry 32(18):4938-4942; Spence, R. A., et al., (1995)Science 267(5200):988-993.}

Furthermore selected B- and/or T-cell epitopes can be added to theGag-polymerase constructs within the deletions of the RT- and INT-codingsequence to replace and augment any epitopes deleted by the functionalmodifications of RT and INT. Alternately, selected B- and T-cellepitopes (including CTL epitopes) from RT and INT can be included in aminimal VLP formed by expression of the synthetic Gag or syntheticGagProt cassette, described above. (For descriptions of known HIV B- andT-cell epitopes see, HIV Molecular Immunology Database CTL SearchInterface; Los Alamos Sequence Compendia, 1987-1997; Internet address:http://hiv-web.lan1.gov/immunology/index.html.)

In another aspect, the present invention comprises Env coding sequencesthat include, but are not limited to, polynucleotide sequences encodingthe following HIV-encoded polypeptides: gp160, gp140, and gp120 (see,e.g., U.S. Pat. No. 5,792,459 for a description of the HIV-1_(SF2)(“SF2”) Env polypeptide). The relationships between these polypeptidesis shown schematically in FIG. 3 (in the figure: the polypeptides areindicated as lines, the amino and carboxy termini are indicated on thegp160 line; the open circle represents the oligomerization domain; theopen square represents a transmembrane spanning domain (TM); and “c”represents the location of a cleavage site, in gp140.mut the “X”indicates that the cleavage site has been mutated such that it no longerfunctions as a cleavage site). The polypeptide gp160 includes the codingsequences for gp120 and gp41. The polypeptide gp41 is comprised ofseveral domains including an oligomerization domain (OD) and atransmembrane spanning domain (TM). In the native envelope, theoligomerization domain is required for the non-covalent association ofthree gp41 polypeptides to form a trimeric structure: throughnon-covalent interactions with the gp41 trimer (and itself), the gp120polypeptides are also organized in a trimeric structure. A cleavage site(or cleavage sites) exists approximately between the polypeptidesequences for gp120 and the polypeptide sequences corresponding to gp41.This cleavage site(s) can be mutated to prevent cleavage at the site.The resulting gp140 polypeptide corresponds to a truncated form of gp160where the transmembrane spanning domain of gp41 has been deleted. Thisgp140 polypeptide can exist in both monomeric and oligomeric (i.e.trimeric) forms by virtue of the presence of the oligomerization domainin the gp41 moiety. In the situation where the cleavage site has beenmutated to prevent cleavage and the transmembrane portion of gp41 hasbeen deleted the resulting polypeptide product is designated “mutated”gp140 (e.g., gp140.mut). As will be apparent to those in the field, thecleavage site can be mutated in a variety of ways. (See, also, WO00/39302).

Wild-type HIV coding sequences (e.g., Gag, Env, Pol, tat, rev, nef, vpr,vpu, vif, etc.) can be selected from any known HIV isolate and thesesequences manipulated to maximize expression of their gene productsfollowing the teachings of the present invention. The wild-type codingregion maybe modified in one or more of the following ways. In oneembodiment, sequences encoding hypervariable regions of Env,particularly V1 and/or V2 were deleted. In other embodiments, mutationswere introduced into sequences, for example, encoding the cleavage sitein Env to abrogate the enzymatic cleavage of oligomeric gp 140

into gp120 monomers. (See, e.g., Earl et al. (1990) PNAS USA 87:648-652;Earl et al. (1991) J. Virol. 65:31-41). In yet other embodiments,hypervariable region(s) were deleted, N-glycosylation sites were removedand/or cleavage sites mutated. As discussed above, different mutationsmay be introduced into the coding sequences of different genes (see,e.g., Table B). For example, Tat coding sequences were modifiedaccording to the teachings of the present specification, for example toaffect the transactivation domain of the gene product (e.g., replacing acystein residue at position 22 with a glycine, Caputo et al. (1996) GeneTherapy 3:235).

To create the synthetic coding sequences of the present invention thegene cassettes are designed to comprise the entire coding sequence ofinterest. Synthetic gene cassettes are constructed by oligonucleotidesynthesis and PCR amplification to generate gene fragments. Primers arechosen to provide convenient restriction sites for subcloning. Theresulting fragments are then ligated to create the entire desiredsequence which is then cloned into an appropriate vector. The finalsynthetic sequences are (i) screened by restriction endonucleasedigestion and analysis, (ii) subjected to DNA sequencing in order toconfirm that the desired sequence has been obtained and (iii) theidentity and integrity of the expressed protein confirmed by SDS-PAGEand Western blotting. The synthetic coding sequences are assembled atChiron Corp. (Emeryville, Calif.) or by the Midland Certified ReagentCompany (Midland, Tex.).

Percent identity to the synthetic sequences of the present invention canbe determined, for example, using the Smith-Waterman search algorithm(Time Logic, Incline Village, Nev.), with the following exemplaryparameters: weight matrix=nuc4×4hb; gap opening penalty=20, gapextension penalty=5, reporting threshold=1; alignment threshold=20.

Various forms of the different embodiments of the present invention(e.g., constructs) may be combined.

Exemplary embodiments of the synthetic polynucleotides of the presentinvention include, but are not limited to, the sequences presented inTable C. TABLE C Type B Synthetic, Codon Optimized Polynucleotides NameFigure Number Description (encoding) GagComplPolmut.SF2 6 Gag complete,RT mutated, (SEQ ID NO: 9) Protease functional; all in frameGagComplPolmutAtt.SF2 7 Gag complete, RT mutated, (SEQ ID NO: 10)Protease attenuated; all in frame GagComplPolmutIna.SF2 8 Gag complete,RT mutated, (SEQ ID NO: 11) Protease non-functional; all in framegagCpolInaTatRevNef.opt_B 9 Gag complete, protease non- (SEQ ID NO: 12)functional, RT mutated, tat mutated, rev mutated, nef mutated; all inframe GagPolmutAtt.SF2 10 Gag, RT mutated, Protease (SEQ ID NO: 13)attenuated; all in frame GagPolmutIna.SF2 11 Gag, RT mutated, Protease(SEQ ID NO: 14) non-functional; all in frame GagProtInaRTmut.SF2 12 Gag,Protease non-functional, (SEQ ID NO: 15) RT mutated; all in frameGagProtInaRTmutTatRevNef.opt_B 13 Gag, protease non-functional, (SEQ IDNO: 16) RT mutated, tat mutated, rev mutated, nef mutated; all in frameGagRTmut.SF2 14 Gag, RT mutated; all in frame (SEQ ID NO: 17)GagTatRevNef.opt_B 15 Gag, tat mutated, rev mutated, (SEQ ID NO: 18) nefmutated; all in frame gp140.modSF162.CwtLmod 16 gp140 derived from SF162(SEQ ID NO: 19) with a HIV Type C (TV1) optimized leader sequencegp140.modSF162.CwtLnat 17 gp140 derived from SF162 (SEQ ID NO: 20) witha HIV Type C (TV1) native leader sequence gp160.modSF162.delV2.mut7 18gp160 derived from SF162, (SEQ ID NO: 21) deletion of V2 loop, mutatedcleavage site gp160.modSF162.delV2.mut8 19 gp160 derived from SF162,(SEQ ID NO: 22) deletion of V2 loop, mutated cleavage siteint.opt.mut.SF2 20 integrase mutated (SEQ ID NO: 23) int.opt.SF2 21integrase (SEQ ID NO: 24) nef.D125G.-myr.opt.SF162 22 nef mutated,myristoyilation (SEQ ID NO: 25) defective nef.D107G.-myr18.opt.SF162 23nef mutated, myristoyilation (SEQ ID NO: 26) defectivenef.opt.D125G.SF162 24 nef mutated (SEQ ID NO: 27) nef.opt.SF162 25 nef(SEQ ID NO: 28) p15RnaseH.opt.SF2 26 p15 RNase H; in-frame (SEQ ID NO:29) p2Pol.opt.YMWM.SF2 27 p2 pol mutated (RT YM, WM) (SEQ ID NO: 30)p2PolInaopt.YM.SF2 28 p2 pol, protease non- (SEQ ID NO: 31) functional,RT YM; all in frame p2Polopt.SF2 29 p2 pol; all in frame (SEQ ID NO: 32)p2PolTatRevNef.opt.native_B 30 p2 pol tat rev nef; all native; (SEQ IDNO: 33) all in frame p2PolTatRevNef.opt_B 31 p2 pol, protease mutated,RT (SEQ ID NO: 34) mutated, tat mutated, rev mutated, nef, mutated; allin frame pol.opt.SF2 32 pol (SEQ ID NO: 35) prot.opt.SF2 33 protease(SEQ ID NO: 36) protIna.opt.SF2 34 protease non-functional (SEQ ID NO:37) protInaRT.YM.opt.SF2 35 protease non-functional, RT (SEQ ID NO: 38)YM mutated; all in frame protInaRT.YMWM.opt.SF2 36 proteasenon-functional, RT (SEQ ID NO: 39) YM WM mutated; all in frameProtInaRTmut.SF2 37 Protease inactive, RT mutated; (SEQ ID NO: 40) allin frame protRT.opt.SF2 38 protease RT; all in frame (SEQ ID NO: 41)ProtRT.TatRevNef.opt_B 39 protease mutated, RT mutated, (SEQ ID NO: 42)tat mutated, rev mutated, nef, mutated; all in frameProtRTTatRevNef.opt_B 40 protease mutated, RT mutated, (SEQ ID NO: 43)tat mutated, rev mutated, nef, mutated; all in framerev.exon1_2.M5-10.opt.SF162 41 rev exon 1 and 2 in-frame, rev (SEQ IDNO: 44) mutated rev.exon1_2.opt.SF162 42 rev exon 1 and 2 in-frame (SEQID NO: 45) RT.opt.SF2 (mutant) 43 RT mutant (SEQ ID NO: 46) RT.opt.SF2(native) 44 RT native (SEQ ID NO: 47) RTmut.SF2 45 RT mutated (SEQ IDNO: 48) tat.exon1_2.opt.C22-37.SF2 46 tat exon 1 and 2 in-frame, tat(SEQ ID NO: 49) mutated tat.exon1_2.opt.C37.SF2 47 tat exon 1 and 2in-frame, tat (SEQ ID NO: 50) mutated TatRevNef.opt.native.SF162 48 tatnative, rev native, nef (SEQ ID NO: 51) native; all in frameTatRevNef.opt.SF162 49 tat mutated, rev mutated, nef (SEQ ID NO: 52)mutated; all in frame TatRevNefGag B 50 tat mutated, rev mutated, nef(SEQ ID NO: 53) mutated, gag; all in frame TatRevNefgagCpolIna B 51 tatmutated, rev mutated, nef (SEQ ID NO: 54) mutated, gag complete,protease non-functional, RT mutated; all in frameTatRevNefGagProtInaRTmut B 52 tat mutated, rev mutated, nef (SEQ ID NO:55) mutated, gag, protease non- functional, RT mutant; all in frameTatRevNefp2Pol.opt_B 53 tat mutated, rev mutated, nef (SEQ ID NO: 56)mutated, p2 pol, protease mutated, RT mutated; all in frameTatRevNefprotRTopt B 54 tat mutated, rev mutated, nef (SEQ ID NO: 57)mutated, protease mutated, RT mutated; all in frame vif.opt.SF2 55optimized vif derived from (SEQ ID NO: 58) SF2 vpr.opt.SF2 56 optimizedvpr derived from (SEQ ID NO: 59) SF2 vpu.opt.SF162 57 optimized vpuderived from (SEQ ID NO: 60) SF162{In Table C, mut or .mut7 or mut 8 envelope mutated in cellular proteasecleavage site between gp120/gp41 (i.e., to prevent cleavage; e.g.,better for purifying protein)}B. Creating Expression Cassettes Comprising the SyntheticPolynucleotides of the Present Invention.

The synthetic DNA fragments of the present invention are cloned into thefollowing expression vectors: pCMVKm2, for transient expression assaysand DNA immunization studies, the pCMVKm2 vector was derived from pCMV6a(Chapman et al., Nuc. Acids Res. (1991) 19:3979-3986) and comprises akanamycin selectable marker, a ColE1 origin of replication, a CMVpromoter enhancer and Intron A, followed by an insertion site for thesynthetic sequences described below followed by a polyadenylation signalderived from bovine growth hormone—the pCMVKm2 vector differs from thepCMV-link vector only in that a polylinker site was inserted intopCMVKm2 to generate pCMV-link; pESN2dhfr and pCMVPLEdhfr (also known aspCMVIII), for expression in Chinese Hamster Ovary (CHO) cells; and,pAcC13, a shuttle vector for use in the Baculovirus expression system(pAcC13, was derived from pAcC12 which was described by Munemitsu S., etal., Mol Cell Biol. 10(11):5977-5982, 1990). See, also co-owned WO00/39302, WO 00/39303, WO 00/39304, and WO 02/04493, for a descriptionof these vectors, all herein incorporated by reference in theirentireties.

Briefly, construction of pCMVPLEdhfr (pCMVIII) was as follows. Toconstruct a DHFR cassette, the EMCV IRES (internal ribosome entry site)leader was PCR-amplified from pCite-4-a+(Novagen, Inc., Milwaukee, Wis.)and inserted into pET-23d (Novagen, Inc., Milwaukee, Wis.) as an Xba-Ncofragment to give pET-EMCV. The dhfr gene was PCR-amplified frompESN2dhfr to give a product with a Gly-Gly-Gly-Ser spacer in place ofthe translation stop codon and inserted as an Nco-BamH1 fragment to givepET-E-DHFR. Next, the attenuated neo gene was PCR amplified from apSV2Neo (Clontech, Palo Alto, Calif.) derivative and inserted into theunique BamH1 site of pET-E-DHFR to give PET-E-DHFR/Neo_((m2)). Then, thebovine growth hormone terminator from pCDNA3 (Invitrogen, Inc.,Carlsbad, Calif.) was inserted downstream of the neo gene to givepET-E-DHFR/Neo_((m2))BGHt. The EMCV-dhfr/neo selectable marker cassettefragment was prepared by cleavage of PET-E-D DHFR/Neo_((m2))BGHt. TheCMV enhancer/promoter plus Intron A was transferred from pCMV6a (Chapmanet al., Nuc. Acids Res. (1991) 19:3979-3986) as a HindIII-Sal1 fragmentinto pUC19 (New England Biolabs, Inc., Beverly, Mass.). The vectorbackbone of pUC19 was deleted from the Nde1 to the Sap1 sites. The abovedescribed DHFR cassette was added to the construct such that the EMCVIRES followed the CMV promoter to produce the final construct. Thevector also contained an ampr gene and an SV40 origin of replication.

Expression vectors of the present invention contain one or more of thesynthetic coding sequences disclosed herein, e.g., shown in the Figures.When the expression cassette contains more than one coding sequence thecoding sequences may all be in-frame to generate one polyprotein;alternately, the more than one polypeptide coding sequences may comprisea polycistronic message where, for example, an IRES is placed 5′ to eachpolypeptide coding sequence.

Example 2 Expression Assays for the Synthetic Coding Sequences

The wild-type sequences are cloned into expression vectors having thesame features as the vectors into which the synthetic HIV-derivedsequences were cloned.

Expression efficiencies for various vectors carrying the wild-type (anyknown isolated) and corresponding synthetic sequence(s) are evaluated asfollows. Cells from several mammalian cell lines (293, RD, COS-7, andCHO; all obtained from the American Type Culture Collection, 10801University Boulevard, Manassas, Va. 20110-2209) are transfected with 2μg of DNA in transfection reagent LT1 (PanVera Corporation, 545 ScienceDr., Madison, Wis.). The cells are incubated for 5 hours in reducedserum medium (Opti-MEM, Gibco-BRL, Gaithersburg, Md.). The medium isthen replaced with normal medium as follows: 293 cells, IMDM, 10% fetalcalf serum, 2% glutamine (BioWhittaker, Walkersville, Md.); RD and COS-7cells, D-MEM, 10% fetal calf serum, 2% glutamine (Opti-MEM, Gibco-BRL,Gaithersburg, Md.); and CHO cells, Ham's F-12, 10% fetal calf serum, 2%glutamine (Opti-MEM, Gibco-BRL, Gaithersburg, Md.). The cells areincubated for either 48 or 60 hours. Supernatants are harvested andfiltered through 0.45 μm syringe filters and, optionally, stored at −20°C.

Supernatants are evaluated using the Coulter p24-assay (CoulterCorporation, Hialeah, Fla., US), using 96-well plates coated with asuitable monoclonal antibody directed against an HIV antigen (e.g., amurine monoclonal directed again an HIV core antigen). The appropriateHIV antigen binds to the coated wells and biotinylated antibodiesagainst HIV recognize the bound antigen. Conjugatedstrepavidin-horseradish peroxidase reacts with the biotin. Colordevelops from the reaction of peroxidase with TMB substrate. Thereaction is terminated by addition of 4N H₂SO₄. The intensity of thecolor is directly proportional to the amount of HIV antigen in a sample.

Chinese hamster ovary (CHO) cells are also transfected with plasmid DNAencoding the synthetic HIV polypeptides described herein (e.g.,pESN2dhfr or pCMVIII vector backbone) using Mirus TransIT-LT1 polyaminetransfection reagent (Pan Vera) according to the manufacturersinstructions and incubated for 96 hours. After 96 hours, media ischanged to selective media (F12 special with 250 μg/ml G418) and cellsare split 1:5 and incubated for an additional 48 hours. Media is changedevery 5-7 days until colonies start forming at which time the coloniesare picked, plated into 96 well plates and screened by Capture ELISA.Positive clones are expanded in 24 well plates and are screened severaltimes for HIV protein production by Capture ELISA, as described above.After reaching confluency in 24 well plates, positive clones areexpanded to T25 flasks (Corning, Corning, N.Y.). These are screenedseveral times after confluency and positive clones are expanded to T75flasks. Positive T75 clones are frozen in LN2 and the highest expressingclones are amplified with 0-5 μM methotrexate (MTX) at severalconcentrations and plated in 100 mm culture dishes. Plates are screenedfor colony formation and all positive closed are again expanded asdescribed above. Clones are expanded an amplified and screened at eachstep capture ELISA. Positive clones are frozen at each methotrexatelevel. Highest producing clones are grown in perfusion bioreactors (3 L,100 L) for expansion and adaptation to low serum suspension cultureconditions for scale-up to larger bioreactors.

Data from experiments performed in support of the present invention showthat the synthetic HIV expression cassettes provided dramatic increasesin production of their protein products, relative to the native(wild-type) sequences, when expressed in a variety of cell lines andthat stably transfected CHO cell lines, which express the desired HIVpolypeptide(s), may be produced. Production of HIV polypeptides usingCHO cells provides (i) correct glycosylation patterns and proteinconformation (as determined by binding to panel of MAbs); (ii) correctbinding to CD4 receptor molecules; (iii) absence of non-mammalian cellcontaminants (e.g., insect viruses and/or cells); and (iv) ease ofpurification.

Example 3 Western Blot Analysis of Expression

Western blot analysis of cells transfected with the HIV expressioncassettes described herein are performed essentially as described inco-owned WO 00/39302. Briefly, human 293 cells are transfected asdescribed in Example 2 with pCMV6a-based vectors containing native orsynthetic HIV expression cassettes. Cells are cultivated for 60 hourspost-transfection. Supernatants are prepared as described. Cell lysatesare prepared as follows. The cells are washed once withphosphate-buffered saline, lysed with detergent [1% NP40 (Sigma ChemicalCo., St. Louis, Mo.) in 0.1 M Tris-HCl, pH 7.5], and the lysatetransferred into fresh tubes. SDS-polyacrylamide gels (pre-cast 8-16%;Novex, San Diego, Calif.) are loaded with 20 μl of supernatant or 12.5μl of cell lysate. A protein standard is also loaded (5 μl, broad sizerange standard; BioRad Laboratories, Hercules, Calif.). Electrophoresisis carried out and the proteins are transferred using a BioRad TransferChamber (BioRad Laboratories, Hercules, Calif.) to Immobilon P membranes(Millipore Corp., Bedford, Mass.) using the transfer buffer recommendedby the manufacturer (Millipore), where the transfer is performed at 100volts for 90 minutes. The membranes are exposed to HIV-1-positive humanpatient serum and immunostained using o-phenylenediamine dihydrochloride(OPD; Sigma).

The results of the immunoblotting analysis are used to show that cellscontaining the synthetic HIV expression cassette produce the expectedHIV-polypeptide(s) at higher per-cell concentrations than cellscontaining the native expression cassette.

Example 4 In Vivo Immunogenicity of Synthetic HIV Expression Cassettes

A. Immunization

To evaluate the immunogenicity of the synthetic HIV expressioncassettes, a mouse study may be performed. The plasmid DNA, e.g.,pCMVKM2 carrying an expression cassette comprising a synthetic sequenceof the present invention, is diluted to the following finalconcentrations in a total injection volume of 100 μl: 20 μg, 2 μg, 0.2μg, and 0.02 μg. To overcome possible negative dilution effects of thediluted DNA, the total DNA concentration in each sample is brought up to20 μg using the vector (pCMVKM2) alone. As a control, plasmid DNAcomprising an expression cassette encoding the native, correspondingpolypeptide is handled in the same manner. Twelve groups of four Balb/cmice (Charles River, Boston, Mass.) are intramuscularly immunized (50 μlper leg, intramuscular injection into the tibialis anterior) usingvarying dosages.

B. Humoral Immune Response

The humoral immune response is checked with a suitable anti-HIV antibodyELISAs (enzyme-linked immunosorbent assays) of the mice sera 0 and 4weeks post immunization (groups 5-12) and, in addition, 6 and 8 weekspost immunization, respectively, 2 and 4 weeks post second immunization(groups 1-4).

The antibody titers of the sera are determined by anti-HIV antibodyELISA. Briefly, sera from immunized mice were screened for antibodiesdirected against an appropriate HIV protein (e.g., HIV p55 for Gag).ELISA microtiter plates are coated with 0.2 μg of HIV protein per wellovernight and washed four times; subsequently, blocking is done withPBS-0.2% Tween (Sigma) for 2 hours. After removal of the blockingsolution, 100 μl of diluted mouse serum is added. Sera are tested at1/25 dilutions and by serial 3-fold dilutions, thereafter. Microtiterplates are washed four times and incubated with a secondary,peroxidase-coupled anti-mouse IgG antibody (Pierce, Rockford, Ill.).ELISA plates are washed and 100 μl of 3, 3′, 5,5′-tetramethyl benzidine(TMB; Pierce) was added per well. The optical density of each well ismeasured after 15 minutes. The titers reported are the reciprocal of thedilution of serum that gave a half-maximum optical density (O.D.).

The results of the mouse immunizations with plasmid-DNAs are used toshow that the synthetic expression cassettes provide improvement ofimmunogenicity relative to the native expression cassettes. Also, thesecond boost immunization induces a secondary immune response after twoweeks (groups 1-3).

C. Cellular Immune Response

The frequency of specific cytotoxic T-lymphocytes (CTL) is evaluated bya standard chromium release assay of peptide pulsed Balb/c mouse CD4cells. HIV protein-expressing vaccinia virus infected CD-8 cells areused as a positive control (vv-protein). Briefly, spleen cells (Effectorcells, E) are obtained from the BALB/c mice (immunized as describedabove). The cells are cultured, restimulated, and assayed for CTLactivity against, e.g., Gag peptide-pulsed target cells as described(Doe, B., and Walker, C. M., AIDS 10(7):793-794, 1996). Cytotoxicactivity is measured in a standard ⁵¹Cr release assay. Target (T) cellsare cultured with effector (E) cells at various E:T ratios for 4 hoursand the average cpm from duplicate wells is used to calculate percentspecific ⁵¹Cr release.

Cytotoxic T-cell (CTL) activity is measured in splenocytes recoveredfrom the mice immunized with HIV DNA constructs described herein.Effector cells from the DNA-immunized animals exhibit specific lysis ofHIV peptide-pulsed SV-BALB (MHC matched) targets cells indicative of aCTL response. Target cells that are peptide-pulsed and derived from anMHC-unmatched mouse strain (MC57) are not lysed. The results of the CTLassays are used to show increased potency of synthetic HIV expressioncassettes for induction of cytotoxic T-lymphocyte (CTL) responses by DNAimmunization.

Example 5 In Vivo Immunogenicity of Synthetic HIV Expression Cassettes

A. General Immunization Methods

To evaluate the immunogenicity of the synthetic HIV expressioncassettes, studies using guinea pigs, rabbits, mice, rhesus macaques andbaboons are performed. The studies are typically structured as follows:DNA immunization alone (single or multiple); DNA immunization followedby protein immunization (boost); DNA immunization followed by Sindbisparticle immunization; immunization by Sindbis particles alone.

B. Guinea Pigs

Experiments may be performed using guinea pigs as follows. Groupscomprising six guinea pigs each are immunized intramuscularly ormucosally at 0, 4, and 12 weeks with plasmid DNAs encoding expressioncassettes comprising one or more the sequences described herein. Theanimals are subsequently boosted at approximately 18 weeks with a singledose (intramuscular, intradermally or mucosally) of the HIV proteinencoded by the sequence(s) of the plasmid boost and/or other HIVproteins. Antibody titers (geometric mean titers) are measured at twoweeks following the third DNA immunization and at two weeks after theprotein boost. These results are used to demonstrate the usefulness ofthe synthetic constructs to generate immune responses, as well as, theadvantage of providing a protein boost to enhance the immune responsefollowing DNA immunization.

C. Rabbits

Experiments may be performed using rabbits as follows. Rabbits areimmunized intramuscularly, mucosally, or intradermally (using a Biojectneedless syringe) with plasmid DNAs encoding the HIV proteins describedherein. The nucleic acid immunizations are followed by protein boostingafter the initial immunization. Typically, constructs comprising thesynthetic HIV-polypeptide-encoding polynucleotides of the presentinvention are highly immunogenic and generate substantial antigenbinding antibody responses after only 2 immunizations in rabbits.

D. Humoral Immune Response

In any immunized animal model, the humoral immune response is checked inserum specimens from the immunized animals with an anti-HIV antibodyELISAs (enzyme-linked immunosorbent assays) at various timespost-immunization. The antibody titers of the sera are determined byanti-HIV antibody ELISA as described above. Briefly, sera from immunizedanimals are screened for antibodies directed against the HIVpolypeptide/protein(s) encoded by the DNA and/or polypeptide used toimmunize the animals. Wells of ELISA microtiter plates are coatedovernight with the selected HIV polypeptide/protein and washed fourtimes; subsequently, blocking is done with PBS-0.2% Tween (Sigma) for 2hours. After removal of the blocking solution, 100 μl of diluted mouseserum is added. Sera are tested at 1/25 dilutions and by serial 3-folddilutions, thereafter. Microtiter plates are washed four times andincubated with a secondary, peroxidase-coupled anti-mouse IgG antibody(Pierce, Rockford, Ill.). ELISA plates are washed and 100 μl of 3, 3′,5,5′-tetramethyl benzidine (TMB; Pierce) was added per well. The opticaldensity of each well is measured after 15 minutes. Titers are typicallyreported as the reciprocal of the dilution of serum that gave ahalf-maximum optical density (O.D.).

Cellular immune response may also be evaluated.

Example 6 DNA-Immunization of Baboons and Rhesus Macaques UsingExpression Cassettes Comprising the Synthetic HIV Polynucleotides of thePresent Invention

A. Baboons

Four baboons are immunized 3 times (weeks 0, 4 and 8) bilaterally,intramuscular into the quadriceps or mucosally using the gene deliveryvehicles described herein. The animals are bled two weeks after eachimmunization and an HIV antibody ELISA is performed with isolatedplasma. The ELISA is performed essentially as described above except thesecond antibody-conjugate is an anti-human IgG, g-chain specific,peroxidase conjugate (Sigma Chemical Co., St. Louis, Md. 63178) used ata dilution of 1:500. Fifty μg/ml yeast extract may be added to thedilutions of plasma samples and antibody conjugate to reducenon-specific background due to preexisting yeast antibodies in thebaboons. Lymphoproliferative responses to are observed in baboons twoweeks post-fourth immunization (at week 14), and enhanced substantiallypost-boosting with HIV-polypeptide (at week 44 and 76). Suchproliferation results are indicative of induction of T-helper cellfunctions.

B. Rhesus Macaques

The improved potency of the synthetic, codon-modified HIV-polypeptideencoding polynucleotides of the present invention, when constructed intoexpression plasmids may be confirmed in rhesus macaques. Typically, themacaques have detectable HIV-specific CTL after two or three 1 mg dosesof modified HIV polynucleotide. In sum, these results demonstrate thatthe synthetic HIV DNA is immunogenic in non-human primates. Neutralizingantibodies may also detected.

Example 7 Co-Transfection of Monocistronic and Multicistronic Constructs

The present invention includes co-transfection with multiple,monocistronic expression cassettes, as well as, co-transfection with oneor more multi-cistronic expression cassettes, or combinations thereof.

Such constructs, in a variety of combinations, may be transfected into293T cells for transient transfection studies.

For example, a bicistronic construct may be made where the codingsequences for the different HIV polypeptides are under the control of asingle CMV promoter and, between the two coding sequences, an IRES(internal ribosome entry site (EMCV IRES); Kozak, M., Critical Reviewsin Biochemistry and Molecular Biology 27(45):385-402, 1992; Witherell,G. W., et al., Virology 214:660-663, 1995) sequence is introduced afterthe first HIV coding sequence and before the second HIV coding sequence.

Supernatants collected from cell culture are tested for the presence ofthe HIV proteins and indicate that appropriate proteins are expressed inthe transfected cells (e.g., if an Env coding sequence was present thecorresponding Env protein was detected; if a Gag coding sequence waspresent the corresponding Gag protein was detected, etc).

The production of chimeric VLPs by these cell lines may be determinedusing electron microscopic analysis. (See, e.g., co-owned WO 00/39302).

Example 8 Accessory Gene Components for an HIV-1 Vaccine: FunctionalAnalysis of Mutated Tat, Rev and Nef Type C Antigens

The HIV-1 regulatory and accessory genes have received increasedattention as components of HIV vaccines due to their role in viralpathogenesis, the high ratio of highly conserved CTL epitopes and theirearly expression in the viral life cycle. Because of various undesirableproperties of these genes, questions regarding their safety andsuitability as vaccine components have been raised. Experimentsperformed in support of the present invention have analyzed candidateHIV-1 subtype C tat, rev, and nef mutants for efficient expression andinactivation of potential deleterious functions. Other HIV subtypeaccessory genes may be evaluated similarly.

Sequence-modified, mutant tat, rev, and nef genes coding for consensusTat, Rev and Nef proteins of South African HIV-1 subtype C wereconstructed using overlapping synthetic oligonucleotides and PCR-basedsite-directed mutagenesis. Constructs of the wild-type genes of theisolates closely resembling the respective consensus sequences were alsomade by PCR. In vitro expression of the constructs was analyzed bywestern blotting. The trans-activation activity of the Tat mutants andnuclear RNA export activity of the Rev mutants were studied aftertransfection of various cell lines using reporter-gene-basedfunctionality assays.

In vitro expression of all constructs was demonstrated by westernblotting using antigen specific mouse serum generated by DNA vaccinationof mice with Tat, Rev, or Nef-expression plasmids. Expression levels ofthe sequence-modified genes were significantly higher than the wild-typegenes.

Subtype B and C Tat cDNA was mutated to get TatC22, TatC37, andTatC22/37. Tat activity assays in three cell lines (RD, HeLa and 293).In the background of the subtype C consensus Tat, a single mutation atC22 was insufficient to inactivate LTR-dependent CAT expression. Incontrast, this activity was significantly impaired in RD, 293 and HeLacells using the single mutation, C37, or the double mutation, C22C37(see Table B). Corresponding results were obtained for Tat mutantsderived from subtype B strains.

Exemplary results are presented in FIG. 4 for transactivation activityof Tat mutants on LTR-CAT plasmid in 293 cells. Three independent assayswere performed for each construct (FIG. 4, legend (1), (2), (3)).

The subtype C constructs TatC22ProtRTTatRevNef and ProtRTTatC22RevNefshowed reduced Tat activity when compared to TatC22 alone, probably dueto structural changes caused by the fusion protein.

For Rev constructs, to test for the loss of function, a CAT assay with areporter plasmid including native or mutated Rev was used. As shown inFIG. 5, compared to wild-type Rev, the mRNA export function of thesubtype C Rev with a double mutation, M5M10 (see Table B), wassignificantly lower. The background levels are shown in the “mock” dataand the pDM128 reporter plasmid without Rev data. Two independent assayswere performed for each construct (FIG. 5, legend (1), (2)).

Assays to measure Nef-specific functions may also be performed (Nefmutations are described in Table B). For example, FACs analysis is usedto look for the presence of MHC1 and CD4 on cell surfaces. Cells areassayed in the presence and absence of Nef expression (for controls), aswell as using the synthetic polynucleotides of the present inventionthat encode native nef protein and mutated nef protein. Down-regulationof MHC1 and CD4 expression indicates that the nef gene product is notfunctional, i.e., if nef is non-functional there is no down regulation.

These data demonstrate the impaired functionality of tat and rev DNAimmunogens that may form part of a multi-component HIV-1 subtype Cvaccine. In contrast to previous published data by other groups, the C22mutation did not sufficiently inactivate the transactivation function ofTat. The C37 mutation appeared to be required for inactivation ofsubtype C and subtype B Tat proteins.

Example 9 Evaluation of Immunogenicity of Various HIV PolypeptideEncoding Plasmids

As noted above, the immunogenicity of any of the polynucleotides orexpression cassettes described herein is readily evaluated. In thefollowing table (Table D) are exemplified procedures involving acomparison of the immunogenicity of subtype B and C envelope plasmids,both individually and as a mixed-subtype vaccine, using electroporation,in rabbits. It will be apparent that such methods are equally applicableto any other HIV polypeptide. TABLE D Total Vol/ Sites/ Grp Animal Imm'n# Adjuvant Immunogen Dose Site Animal Route 1 1-4 1, 2 — pCMV 160 TV1DNA 1.0 mg 0.5 ml 2 IM/Quad (Electro) 3 — pCMV 160 TV1 DNA 1.0 mg 0.5 ml2 IM/Quad (Electro) MF59C Protein TBD 0.05 mg  0.5 ml 2 IM/Glut 2 5-8 1,2 — pCMV 160 dV2 TV1 1.0 mg 0.5 ml 2 IM/Quad DNA (Electro) 3 — pCMV 160dV2 TV1 1.0 mg 0.5 ml 2 IM/Quad DNA (Electro) MF59C Protein TBD 0.05 mg 0.5 ml 2 IM/Glut 3  9-12 1, 2 — pCMV 160 dV1/V2 1.0 mg 0.5 ml 2 IM/QuadTV1 DNA (Electro) 3 — pCMV 160 dV1/V2 1.0 mg 0.5 ml 2 IM/Quad TV1 DNA(Electro) MF59C Protein TBD 0.05 mg  0.5 ml 2 IM/Glut 4 13-16 1, 2 —pCMV 140 TV1 DNA 1.0 mg 0.5 ml 2 IM/Quad (Electro) 3 — pCMV 140 TV1 DNA1.0 mg 0.5 ml 2 IM/Quad (Electro) MF59C Protein TBD 0.05 mg  0.5 ml 2IM/Glut 5 17-20 1, 2 — pCMV140dV2TV1 1.0 mg 0.5 ml 2 IM/Quad DNA(Electro) 3 — pCMV140dV2TV1 1.0 mg 0.5 ml 2 IM/Quad DNA (Electro) MF59CProtein TBD 0.05 mg  0.5 ml 2 IM/Glut 6 21-24 1, 2 — pCMV 140 dV1/V2 1.0mg 0.5 ml 2 IM/Quad TV1 DNA (Electro) 3 — pCMV 140 dV1/V2 1.0 mg 0.5 ml2 IM/Quad TV1 DNA (Electro) MF59C Protein TBD 0.05 mg  0.5 ml 2 IM/Glut7 25-28 1, 2 — pSIN140dV2SF162 1.0 mg 0.5 ml 2 IM/Quad DNA (Electro) 3 —pSIN 140 dV2 SF162 1.0 mg 0.5 ml 2 IM/Quad DNA (Electro) MF59C ProteinTBD 0.05 mg  0.5 ml 2 IM/Glut 8 29-32 1, 2 — pCMV 140 dV2 SF162 1.0 mg0.5 ml 2 IM/Quad DNA (Electro) 3 — pCMV 140 dV2 SF162 1.0 mg 0.5 ml 2IM/Quad DNA (Electro) MF59C Protein TBD 0.05 mg  0.5 ml 2 IM/Glut 933-36 1, 2 — pCMV 140 Q154 1.0 mg 0.5 ml 2 IM/Quad SF162 DNA (Electro) 3— pCMV 140 Q154 1.0 mg 0.5 ml 2 IM/Quad SF162 DNA (Electro) MF59CProtein TBD 0.05 mg  0.5 ml 2 IM/Glut 10 37-40 1, 2 — pCMV 140 dV2 SF1621.0 mg 0.5 ml 2 IM/Quad DNA (Electro) pCMV 140 dV2 TV1 1.0 mg DNA 3 —pCMV 140 dV2 SF162 1.0 mg 0.5 ml 2 IM/Quad DNA (Electro) pCMV 140 dV2TV1 1.0 mg DNA MF59C Protein TBD 0.05 mg  0.5 ml 2 IM/Glut 11 41-44 —pCMV 140 dV2 SF162 1.0 mg 0.5 ml 2 IM/Quad DNA (Electro) pCMV 140 dV2TV1 1.0 mg DNA 1, 2 — pCMV 140 dV2 SF162 1.0 mg 0.5 ml 2 IM/Quad DNA(Electro) pCMV 140 dV2 TV1 1.0 mg DNA 3 MF59C Protein TBD 0.05 mg  0.5ml 2 IM/Glut

The MF59C adjuvant is a microfluidized emulsion containing 5% squalene,0.5% tween 80, 0.5% span 85, in 10 mM citrate pH 6, stored in 10 mLaliquots at 4° C.

Immunogens are prepared as described in the following table (Table E)for administration to animals in the various groups. Concentrations mayvary from those described in the table, for example depending on thesequences and/or proteins being used. TABLE E Group Preparation 1-9Immunization 1-3: pCMV and pSIN based plasmid DNA in Saline +Electroporation Subtype B and C plasmids will be provided frozen at aconcentration of 1.0 mg/ml in sterile 0.9% saline. Store at −80° C.until use. Thaw DNA at room temperature; the material should be clear orslightly opaque, with no particulate matter. Animals will be shavedprior to immunization, under sedation of 1x dose IP (by animal weight)of Ketamine-Xylazine (80 mg/ml-4 mg/ml). Immunize each rabbit with 0.5ml DNA mixture per side (IM/Quadriceps), 1.0 ml per animal. Follow theDNA injection with Electroporation using a 6-needle circular array with1 cm diameter, 1 cm needle length. Electroporation pulses were given at20 V/mm, 50 ms pulse length, 1 pulse/s. Immunization 3: ProteinImmunization Proteins will be provided at 0.1 mg/ml in citrate buffer.Store at −80° C. until use. Thaw at room temperature; material should beclear with no particulate matter. Add equal volume of MF59C adjuvant tothawed protein and mix well by inverting the tube. Immunize each rabbitwith 0.5 ml adjuvanted protein per side, IM/Glut for a total of 1.0 mlper animal. Use material within 1 hour of the addition of adjuvant.Immunization 1-3: Combined subtype B and C plasmid DNA in Saline Theimmunogen will be provided at 2.0 mg/ml total DNA (1 mg/ml of eachplasmid) in sterile 0.9% saline. Store at −80° C. until use. Thaw DNA atroom temperature; the material should be clear or slightly opaque, withno particulate matter. Animals will be shaved prior to immunization,under sedation of 1x dose IP (by animal weight) of Ketamine-Xylazine (80mg/ml-4 mg/ml). Immunize each rabbit with 0.5 ml DNA mixture per side(IM/Quadriceps), 1.0 ml per animal. Follow the DNA injection withElectroporation using a 6-needle circular array with 1 cm diameter, 1 cmneedle length. Electroporation pulses were given at 20 V/mm, 50 ms pulselength, 1 pulse/s. 10-11 Immunization 3: Protein Immunization Proteinswill be provided at 0.1 mg/ml in citrate buffer. Store at −80° C. untiluse. Thaw at room temperature; material should be clear with noparticulate matter. Add equal volume of MF59C adjuvant to thawed proteinand mix well by inverting the tube. Immunize each rabbit with 0.5 mladjuvanted protein per side, IM/Glut for a total of 1.0 ml per animal.Use material within 1 hour of the addition of adjuvant.

The immunization (Table F) and bleeding (Table G) schedules are asfollows: TABLE F Imm'n: 1 2 3 3 Weeks: Group 0 4 16 16 1 pCMV 160 TV1DNA pCMV 160 TV1 DNA pCMV 160 TV1 DNA Protein + MF59C 2 pCMV 160 dV2 TV1DNA pCMV 160 dV2 TV1 DNA pCMV 160 dV2 TV1 DNA Protein + MF59C 3 pCMV 160dV1/V2 TV1 DNA pCMV 160 dV1/V2 TV1 DNA pCMV 160 dV1/V2 TV1 DNA Protein +MF59C 4 pCMV 140 TV1 DNA pCMV 140 TV1 DNA pCMV 140 TV1 DNA Protein +MF59C 5 pCMV 140 dV2 TV1 DNA pCMV 140 dV2 TV1 DNA pCMV 140 dV2 TV1 DNAProtein + MF59C 6 pCMV 140 dV1/V2 TV1 DNA pCMV 140 dV1/V2 TV1 DNA pCMV140 dV1/V2 TV1 DNA Protein + MF59C 7 pSIN 140 dV2 SF162 DNA pSIN 140 dV2SF162 DNA pSIN 140 dV2 SF162 DNA Protein + MF59C 8 pCMV 140 dV2 SF162DNA pCMV 140 dV2 SF162 DNA pCMV 140 dV2 SF162 DNA Protein + MF59C 9 pCMV140 Q154 SF162 DNA pCMV 140 Q154 SF162 DNA pCMV 140 Q154 SF162 DNAProtein + MF59C 10 pCMV 140 dV2 SF162 DNA + pCMV pCMV 140 dV2 SF162DNA + pCMV pCMV 140 dV2 SF162 DNA + pCMV Protein + MF59C 140 dV2 TV1 DNA140 dV2 TV1 DNA 140 dV2 TV1 DNA 11 pCMV 140 dV2 SF162 DNA + pCMV pCMV140 dV2 SF162 DNA + pCMV pCMV 140 dV2 SF162 DNA + pCMV Protein + MF59C140 dV1/V2 TV1 DNA 140 dV1/V2 TV1 DNA 140 dV1/V2 TV1 DNA

TABLE G Bleed: 0 1 2 3 4 5 6 7 8 9 10 Week: −3 4 6 8 12 16 18 20 24 28TBD Sample: Clotted Clotted Clotted Clotted Clotted Clotted ClottedClotted Clotted Bld. Clotted Bld. Clotted Bld. Bld. Bld. Bld. Bld. Bld.Bld. Bld. Bld. for Serum for Serum for Serum for Serum for Serum forSerum for Serum for Serum for Serum for Serum for Serum Volume: 20 cceach 20 cc each 20 cc each 20 cc each 20 cc each 20 cc each 20 cc each20 cc each 20 cc each 20 cc each 20 cc each Method: AA/MEV AA/MEV AA/MEVAA/MEV AA/MEV AA/MEV AA/MEV AA/MEV AA/MEV AA/MEV CP

Example 10 Mice Immunization Studies with Gag and Pol Constructs

Cellular and Humoral immune responses were evaluated in mice(essentially as described in Example 4) for the following constructs:Gag, GagProtease(+FS) (GP1, protease codon optimized and inactivation ofINS; GP2, protease only inactivation of INS), GagPolΔintegrase withframeshift (gagFSpol), and GagPolΔintegrase in-frame (GagPol) (see FIG.63). Versions of GagPolΔintegrase in-frame were also designed withattenuated (GagPolAtt) or non-functional Protease (GagPolIna).

In vitro expression data showed comparable expression of p55Gag andp66RT using Gag alone, GagProtease(+FS), GagFSpol and GagPolIna.Constructs with fully functional or attenuated protease (GagPol orGagPolAtt) were less efficient in expression of p55Gag and p66RT,possibly due to cytotoxic effects of protease.

DNA immunization of mice using Gag vs. GP1 and GP2 in pCMV vectors wasperformed intramuscularly in the tibialis anterior. Mice were immunizedat the start of the study (0 week) and 4 weeks later. Bleeds wereperformed at 0, 4, and 6 weeks. DNA doses used were as follows: 20 μg, 2μg, 0.2 μg, and 0.02 μg.

DNA immunization of mice using Gag vs. gagFSpol in pCMV vectors wasperformed intramuscularly in the tibialis anterior. Mice were immunizedat the start of the study (0 week) and challenged 4 weeks later withrecombinant vaccinia virus encoding Gag (rVVgag). Bleeds were performedat 0 and 4 weeks. DNA doses used were as follows: 20 μg, 2 μg, 0.2 μg,and 0.02 μg.

DNA immunization of mice using Gag vs. gagFSpol and gagpol in pCMVvectors was performed intramuscularly in the tibialis anterior. Micewere immunized at the start of the study (0 week) and challenged 4 weekslater with recombinant vaccinia virus encoding Gag (rVVgag). Bleeds wereperformed at 0 and 4 weeks. DNA doses used were as follows: 2 μg, 0.2μg, 0.02 μg, and 0.002 μg.

Cellular immune responses against Gag were comparable for all testedvariants, for example, Gag, GagProtease, gagFSpol and GagPolIna all hadcomparable potencies.

Humoral immune responses to Gag were also comparable with the exceptionof GP2 and especially GP1. Humoral immune responses were weaker inconstructs comprising functional or attenuated proteases which may bedue to less efficient secretion of p55Gag caused by overactive protease.

In vitro and in vivo experiments, performed in support of the presentinvention, suggest that the expression and immunogenicity of Gag wascomparable with all constructs. Exceptions were GagPol in-frame withfully functional or attenuated protease. This may be the result ofcytotoxic effects of protease. The immune response in mice correlatedwith relative levels of expression in vitro.

Further, plasmid DNA vaccines containing synthetic sequence-modifiedgene cassettes for high-level expression of HIV-1 subtype C Gag and Polwere produced. Sequences were selected from a Botswanan HIV-1 strainbased on its homology with other subtype C isolates. Numerous genecassettes were constructed that expressed Gag and Pol alone or GagPol.In some constructs, the frameshift between Gag and Pol coding sequenceswas removed to increase Pol expression. Deletions and mutations wereintroduced into the Pol to inactivate integrase, protease, and reversetranscriptase activities. In vitro analyses included transienttransfections followed by p24 antigen capture ELISA and Western-blottingof supernatants and cell lysates. Two different mouse strains (CB6F1 andBalb/c) were then immunized with titrated doses of plasmid DNA rangingfrom 20 μg to 2 ng per dose. Humoral and cellular immune responses weremeasured by antibody ELISA and intracellular cytokine staining,respectively.

All constructs expressed proteins of the expected sizes as determined byWestern blot. Gag-specific CD8+ T-cell responses were measured in CB6F1mice immunized with either Gag or GagPol DNA vaccines followingstimulation of spleen cells using a previously identified Gag-specificpeptide epitope. Pol-specific responses were measured in immunizedBalb/c mice after screening 236 (12-15 mer) overlapping peptides; atleast 4 novel peptide epitopes were identified. The levels of CD8+ IFN+cells observed after immunization at different doses were comparable foreither the single gene (alone or mixed) or the multigenic GagPolcassettes. A positive response could be detected with as little as 2 ngof DNA.

Example 11 Protein Expression Immunogenicity, and Generation ofNeutralizing Antibodies Using Type C Derived Envelope Polypeptides

Envelope (Env) vaccines derived from the subtype C primary isolate, TV1,recovered from a South African individual, were tested in rabbits asfollows. Gene cassettes were designed to express the gp120 (surfaceantigen), gp140 (surface antigen plus ectodomain of transmembraneprotein, gp41), and full-length (gp120 plus gp41) gp 160 forms of theHIV-1 envelope polyprotein with and without deletions of the variableloop regions, V2 and V1V2. All of the genes were sequence-modified toenhance expression of the encoded Env glycoproteins in a Rev-independentfashion and they were subsequently cloned into pCMV-based plasmidvectors for DNA vaccine and protein production applications as describedabove. The sequences were codon optimized as described herein. Briefly,all the modified envelope genes were cloned into the Chiron pCMVlinkplasmid vector, preferably into EcoRI/XhoI sites.

A. Protein Expression

Full-length (gp160), truncated gp140 (Env ectodomain only) and gp120native versions of the TV1 Env antigen were produced from the expressioncassettes described herein. The gp140 encoding sequences weretransiently transfected into 293T cells. The expression levels of thegene products were evaluated by an in-house antigen capture ELISA.Envelope genes constructed from the native sequences of TV001c8.2,TV001c8.5 and TV002c12.1 expressed the correct proteins in vitro, withgp140TV001c8.2 exhibiting the highest level of expression. In addition,the Env protein expressed from the TV1-derived clone 8.2 was found tobind the CD4 receptor protein indicating that this feature of theexpressed protein is maintained in a functional conformation. Thereceptor binding properties/functionality of the expressed TV1 gp 160protein result was also confirmed by a cell-fusion assay.

Total expression increased approximately 10-fold for synthetic gp 140constructs compared with the native gp140 gene cassettes. Both themodified gp120 and gp140 variants secreted high amounts of protein inthe supernatant. In addition, the V2 and V1V2 deleted forms of gp140expressed approximately 2-fold more protein than the intact gp140.Overall, the expression levels of synthetic gp140 gene variantsincreased 10 to 26-fold compared with the gp140 gene with nativesequences.

In sum, each synthetic construct tested showed more than 10-foldincreased levels of expression relative to those using the native codingsequences. Moreover, all expressed proteins were of the expectedmolecular weights and were shown to bind CD4. Stable CHO cell lines werederived and small-scale protein purification methods were used toproduce small quantities of each of the undeleted and V-deletedoligomeric forms (o-gp140) of these proteins for vaccine studies.

B. Neutralization Properties of TV001 and TV002 Viral Isolates

The transient expression experiment showed that the envelope genesderived from the TV001 and TV002 virus isolates expressed the desiredprotein products. Relative neutralization sensitivities of these twoviral strains using sera from 18 infected South African individuals(subtypes B and C) were as follows. At a 1:10 serum dilution, the TV2strain was neutralized by 18 of 18 sera; at 1:50, 16 of 18; at 1:250,15/18. In comparison, the TV1 isolate was neutralized by 15 of 18 at1:10; only 6 of 18 at 1:50; and none of the specimens at 1:250. Inaddition, the TV001 patient serum showed neutralization activity againstthe TV002 isolate at all dilutions tested. In contrast, the TV002 showedneutralization of TV001 only at the 1:10 serum dilution. These resultssuggest that TV001 isolate is capable of inducing a broader and morepotent neutralizing antibody response in its infected host than TV002.

C. Immunogenicity of the Modified TV1 Env DNA and Protein Antigens inRabbit Studies

TV1 Env DNA (comprising the synthetic expression cassettes) and proteinvaccines were administrated as shown in the following Table H. TABLE HGroups Plasmid DNA (0, 4, and 20 wks) Protein boost (20 wks) 1pCMVgp160.TV1 o-gp140.TV1 2 pCMVgp160dV2.TV1 o-gp140dV2.TV1 3pCMVgp160dV1V2.TV1 o-gp140dV1V2.TV1 4 pCMVgp140.TV1 o-gp140.TV1 5pCMVgp140dV2.TV1 o-gp140dV2.TV1 6 pCMVgp140dV1V2.TV1 o-gp140dV1V2.TV1 7pCMVgp140dV2.SF162 o-gp140dV2.SF162

Seven groups of 4 rabbits per group were immunized with the designatedplasmid DNA and oligomeric Env protein antigens. Three doses of DNA, 1mg of DNA per animal per immunization, were administratedintramuscularly by needle injection followed by electroporation on weeks0, 4, and 20 weeks. A single dose of 100 ug of Env protein in MF59adjuvant also was given intramuscularly in a separate site at 20 weeks.

The DNA immunization used subtype C sequence-modified genes (TV1)—gp160,gp160dV2, gp160dV1V2, gp140, gp140dV2 and gp140dV1V2—as well as asubtype B SF162 sequence modified gp140dV2. DNA immunizations wereperformed at 0, 4, and 20 weeks by needle injection by the intramuscularroute using electroporation to facilitate transfection of the musclecells and of resident antigen presenting cells.

A single Env protein booster (in MF59 adjuvant) was given at 20 weeks byintramuscular injection at a separate site. Antibody titers wereevaluated by ELISA following each successive immunization. Serumspecimens were collected at 0, 4, 6, 8, 12, 22, and 24 weeks. Serumantibody titers were measured on ELISA. 96-well plates were coated witha protein in a concentration of 1 ug/ml. Serum samples were dilutedserially 3-fold. Goat anti-rabbit peroxidase conjugate (1:20,000) wasused for detection. TMB was used as the substrate, and the antibodytiters were read at 0.6 OD at 450 nm.

Neutralizing antibody responses against PBMC-grown R5HIV-1 strains weremonitored in the sera collected from the immunized rabbits using twodifferent assays in two different laboratories, the 5.25 reportercell-line based assay at Chiron and the PBMC-based assay of DavidMontefiori at Duke University. Results are shown in FIGS. 66, 67, and68. The Chiron assay was conducted essentially as follows. Neutralizingantibody responses against the PBMC-grown subtype C TV001 and TV002strains were measured using an in-house reporter cell line assay thatuses the 5.25 cell line. This cell has CD4, CCR5, CXCR4 and BONZOreceptor/co-receptors on its cell membrane. The parental CEM cell linewas derived from a 4-year-old Caucasian female with acute lymphoblasticleukemia, which was fused with the human B cell line 721.174, creatingCEM×174. LTR-GFP was transfected into the cells after the CCR5 gene(about 1.1 kb) was cloned into the BamH-I (5′) and Sal-I (3′) of thepBABE puro retroviral vector, and subsequently introduced into theCEM×174. The green fluorescence protein (GFP) of the cells was detectedby flow cytometer (FACScan). For the virus neutralization assay, 50 ulof titrated virus and 50 ul of diluted immune or pre-immune serum wereincubated at room temperature for one hour. This mixture was added intowells with 10⁴/ml cells plated in a 24 well plate, and incubated at 37°C. for 5 to 7 days. The cells were then fixed with 2% of formaldehydeafter washing with PBS. Fifteen thousand events (cells) were collectedfor each sample on a Becton Dickinson FACScan using Cellquest software.The data presented were the mean of the triplicate wells. The percentneutralization was calculated compared to the virus control using thefollowing equation: % virus Inhibition=(viruscontrol-experimental)/(virus control −cell control)×100. Any virusinhibition observed in the pre-bleed has been subtracted for eachindividual animal. Values>50% are considered positive and arehighlighted in gray.

In FIG. 67, the “#” indicates that animals had high levels of virusinhibition in pre-bleed serum (>20% virus inhibition) that impacted themagnitude of the observed inhibition and in some cases, our ability toscore the serum as a positive or negative for the presence ofsignificant neutralizing antibody activity (<50% inhibition).

For the data presented in FIG. 68, serum samples were collected after asingle protein boost (post-third) were screened in triplicate at a 1:8dilution with virus (1:24 after addition of cells). Values shown are the% reduction in p24 synthesis relative to that in the correspondingpre-bleed control samples. Zero values indicate no or negative valueswere measured. NV, not valid due to virus inhibition in pre-immuneserum. Neutralization was considered positive when p24 was reduced by atleast 80%; these samples are highlighted in dark gray. Sample withlighter gray shading showed at least a 50% reduction in p24 synthesis.

FIG. 64 shows the ELISA data when plates were coated with the monomericgp120.TV1 protein. This protein is homologous to the subtype C genesused for the immunization. All immunization groups produced highantibody titers after the second DNA immunization. The groups immunizedwith gp140 forms of DNA have relatively higher geometric mean antibodytiters as compared to the groups using gp160 forms after both first andsecond DNA immunizations. Both the gp140.TV1 and gp140dV1V2.TV1 genesproduced high antibody titers at about 10⁴ at two weeks post second DNA;the gp140dV2.TV1 plasmid yielded the highest titers of antibodies (>104)at this time point and all others. The binding antibody titers to thegp120.TV1 protein were higher for the group immunized with thehomologous gp140dV2.TV1 genes than that with the heterologousgp140dV2.5F162 gene which showed titers of about 10³. All the groups,showed some decline in antibody titers by 8 weeks post the second DNAimmunization. Following the DNA plus protein booster at 20 weeks, allgroups reached titers above that previously observed after the secondDNA immunization (0.5-1.0 log increases were observed). After theprotein boost, all animals receiving the o-gp140dV2.TV1 protein whetherprimed by the gp140dV2.TV1 or gp160dV2.TV1 DNA, showed the highest Abtiters.

Binding antibody titers were also measured using ELISA plates coatedwith either oligomeric subtype C o-gp140dV2.TV1 or subtype Bo-gp140dV2.5F162 proteins (FIG. 65). For all the TV1 Env immunizedgroups, the antibody titers measured using the oligomeric protein,o-gp140dV2.TV1 were higher than those measured using the monomeric(non-V2-deleted) protein, gp120.TV1. In fact, for these groups, thetiters observed with the heterologous subtype B o-gp140dV2.5F162 proteinwere comparable to or greater than those measured with the subtype C TV1gp120. Nevertheless, all groups immunized with subtype C immunogensshowed higher titers binding to the subtype C o-gp140dV2.TV1 proteinthan to the subtype B protein gp140dV2.5F162. Conversely, the groupimmunized with the gp140dV2.5F162 immunogen showed higher antibodytiters with the oligomeric subtype B protein relative its subtype Ccounterpart. Overall, all three assays demonstrated that high antibodycross-reactive antibodies were generated by the subtype CTV1-based DNAand protein immunogens.

The results indicate that the subtype C TV1-derived Env DNA and proteinantigens are immunogenic inducing high titers of antibodies in immunizedrabbits and substantial evidence of neutralizing antibodies against bothsubtype B and subtype C R5 virus strains. In particular, thegp140dV2.TV1 antigens have induced consistent neutralizing responsesagainst the subtype B SF162EnvDV2 and subtype C TV2 strains. Thus,TV1-based Env DNA and protein-based antigens are immunogenic and inducehigh titer antibody responses reactive with both subtype C and subtype BHIV-1 Env antigens. Neutralizing antibody responses against theneutralization sensitive subtype B R5HIV-1_(SF162DV2) strain wereobserved in some groups after only two DNA immunizations. Following asingle booster immunization with Env protein, the majority of rabbits ingroups that received V2-deleted forms of the TV1 Env showedneutralization activity against the closely related subtype C TV2primary strain.

Example 12 Immunological Responses in Rhesus Macaques

Cellular and humoral immune responses were evaluated in three groups ofrhesus macaques (each group was made up of four animals) in animmunization study structured as shown in Table I. The route ofadministration for the immunizing composition was electroporation ineach case. Antibody titers are shown in Table I for two weekspost-second immunization. TABLE I Formulation of Immunizing GroupComposition* Animal # Titer 1 pCMVgag (3.5 mg) + pCMVenv A 3325 (2.0 mg)B 4000 C (previously 1838 immunized with HCV core ISCOMS, rVVC core E1)D (previously 1850 immunized with HCV core ISCOMS, rVVC core E1) 2pCMVgag (3.5 mg) + pCMVpol A (previously 525 (4.2 mg) immunized with HCVcore ISCOMS, rVVC core E1, p55gag_(LAI)(VLP)) B 5313 C 6450 D 5713 3pCMVgag-pol (5.0 mg) A (previously 0 immunized with HCV core ISCOMS,rVVC core E1, pCMVgagSF2) B (previously 1063 immunized with rVVC/E1,pCMV Epo-Epi, HIV/HCV-VLP, pCMVgagSF2, pUCgp120 SF2) C 513 D (previously713 immunized with rVVC/E1, HIV/HCV-VLP)*pCMVgag = pCMVKm2.GagMod Type C BotswanapCMVenv = pCMVLink.gp140env.dV2.TV1 (Type C)pCMVpol = pCMVKm2.p2Pol.mut.Ina Type C BotswanapCMVgag-pol = pCMVKm2.gagCpol.mut.Ina Type C Botswana

Pre-immune sera were obtained at week 0 before the first immunization.The first immunization was given at week 0. The second immunization wasgiven at week 4. The first bleed was performed at 2 weeks post-secondimmunization (i.e., at week 6). A third immunization will be given atweek 8 and a fourth at week 16. Animals 2A, 3A, 3B and 3D had beenvaccinated previously (approximately 4 years or more) with gag plasmidDNA or gag VLP (subtype B).

Bulk CTL, ⁵¹Cr-release assays, and flow cell cytometry methods were usedto obtain the data in Tables J and K. Reagents used for detecting gag-and pol-specific T-cells were (i) synthetic, overlapping peptidesspanning “gagCpol” antigen (n=377), typically the peptides were pools of15-mers with overlap by 11, the pools were as follows, pool 1, n=1-82,pool 2, n=83-164, pool 3, n=165-271, pool 4, n=272-377, accordinglypools 1 and 2 are “gag”-specific, and pools 3 and 4 are “pol”-specific,and (ii) recombinant vaccinia virus (rVV), for example, rVVgag965,rVVp2Pol975 (contains p2p7gag975), and VV_(wr)parent.

Gag-specific IFNγ+CD8+T-cells, Gag-specific IFNγ+CD4+T-cells,Pol-specific IFNγ+CD8+T-cells, and Pol-specific IFNγ+CD4+T-cells inblood were determined for each animal described in Table I above, postsecond immunization. The results are presented in Tables J and K. It ispossible that some of the pol-specific activity shown in Table K wasdirected against p2p7gag. TABLE J Gag Assay Results Gag Specific CD8+Gag Specific CD4+ Responses Responses Group/ Immunizing LPA(SI) Flow CTLFlow Animal Composition p55 Pool 1 Pool 2 IFNg+ Pool 1 Pool 2 IFNg+ 1ApCMVgag 3.3 5.9 3.8 496 minus minus 225 pCMVenv 1B pCMVgag 11.8 4.4 1.5786 minus minus 160 pCMVenv 1C pCMVgag 5.7 1.1 2.4 361 plus plus 715pCMVenv 1D pCMVgag 6.5 3.1 1.6 500 plus ? 596 pCMVenv 2A pCMVgag 4.8 4.81.6 405 plus minus 1136 pCMVpol 2B pCMVgag 12.5 6.8 3.3 1288 plus minus2644 pCMVpol 2C pCMVgag 6.0 3.8 2.1 776 minus minus 0 pCMVpol 2D pCMVgag18.9 13.5 5.4 1351 minus minus 145 pCMVpol 3A pCMV 12.2 7.0 1.5 560 plusplus 3595 gagpol 3B pCMV 2.7 5.6 1.3 508 plus ? 3256 gagpol 3C pCMV 11.65.0 1.2 289 minus ? 617 gagpol 3D pCMV 1.5 1.2 1.4 120 minus minus 277gagpol? = might be positive on rVVp2Pol.

TABLE K Pol Assay Results Pol Specific Pol Specific CD8+ CD4+ ResponseResponses Immunizing LPA(SI) Flow CTL Flow Group/Animal Composition Pool3 Pool 4 IFNg+ Pool 3 Pool 4 IFNg+ 1A pCMVgag 1 1.2 0 minus minus 0pCMVenv 1B pCMVgag 1 1 0 minus minus 0 pCMVenv 1C pCMVgag 1 1.1 0 minusminus 0 pCMVenv 1D pCMVgag 1.2 1.3 0 minus minus 262 pCMVenv 2A pCMVgag1.1 0.9 92 minus minus 459 pCMVpol 2B pCMVgag 2.5 1.8 107 minus minus838 pCMVpol 2C pCMVgag 1.2 1.1 52 plus minus 580 pCMVpol 2D pCMVgag 2.52.7 113 plus plus 5084 pCMVpol 3A pCMV 2.7 2.4 498 minus minus 3631gagpol 3B pCMV 1.1 1 299 minus minus 1346 gagpol 3C pCMV 2.1 1.4 369minus minus 399 gagpol 3D pCMV 1.3 1.8 75 minus minus 510 gagpol

Further, based on the mice data (Example 10), groups of rhesus macaqueswere immunized with the selected Gag, Gag+Pol, or GagPol constructs.Table L shows anti-gag and anti-pol CD4 and CD8 responses in macaquesusing various doses of DNA after one or multiple doses with various gagand pol constructs (e.g., subtype C gag, gagpol, gag and pol separately,gagcpol, gagpolIna, etc.) DNA doses varied from 0.002 μg, 0.02 μg, 0.2μg, 20 μg, 3.5 mg. TABLE L CD4 response CD8 response DNA anti-gaganti-pol anti-gag anti-pol gag plus minus plus minus gag + pol plus plusplus plus gagpol plus plus plus plus

These results support that the constructs of the present invention arecapable of generating specific cellular and humoral responses againstthe selected HIV-polypeptide antigen(s).

Although preferred embodiments of the subject invention have beendescribed in some detail, it is understood that obvious variations canbe made without departing from the spirit and the scope of the inventionas defined by the appended claims.

1. A nucleic acid comprising an expression cassette, wherein theexpression cassette comprises a polynucleotide sequence encoding apolypeptide including a polypeptide selected from the group consistingof: (a) an HIV Gag polypeptide, wherein the polynucleotide sequenceencoding said Gag polypeptide comprises a sequence having at least 90%sequence identity to the full length of a sequence selected from thegroup consisting of SEQ ID NO:9; SEQ ID NO:10; SEQ ID NO:11; SEQ IDNO:12; and SEQ ID NO:16; (b) an HIV Gag polypeptide, wherein thepolynucleotide sequence encoding said Gag polypeptide comprises asequence having at least 98% sequence identity to the full length of asequence selected from the group consisting of SEQ ID NO:13; SEQ IDNO:14; SEQ ID NO:15; and SEQ ID NO:55; (c) an HIV Gag polypeptide,wherein the polynucleotide sequence encoding said Gag polypeptidecomprises a sequence having at least 95% sequence identity to the fulllength of SEQ ID NO:17; (d) an HIV Nef polypeptide, wherein thepolynucleotide sequence encoding said Nef polypeptide comprises asequence having at least 90% sequence identity to the full length of asequence selected from the group consisting of SEQ ID NO:25; SEQ IDNO:26; SEQ ID NO:27; SEQ ID NO:28; SEQ ID NO:33 and SEQ ID NO:34; (e) anHIV Prot polypeptide, wherein the polynucleotide sequence encoding saidProt polypeptide comprises a sequence having at least 98% sequenceidentity to the full length of SEQ ID NO:39; (f) an HIV Tat polypeptide,wherein the polynucleotide sequence encoding said Tat polypeptidecomprises a sequence having at least 90% sequence identity to the fulllength of a sequence selected from the group consisting of SEQ ID NO:42;SEQ ID NO:43; SEQ ID NO:51; SEQ ID NO:53; SEQ ID NO:54; SEQ ID NO:56;and SEQ ID NO:57; (g) an HIV Rev polypeptide, wherein the polynucleotidesequence encoding said Rev polypeptide comprises a sequence having atleast 90% sequence identity to the full length of SEQ ID NO:44 or SEQ IDNO:45; (h) an HIV Vif polypeptide, wherein the polynucleotide sequenceencoding said Vif polypeptide comprises a sequence having at least 90%sequence identity to at least 30 contiguous base pairs of SEQ ID NO:58;(i) an HIV Vpr polypeptide, wherein the polynucleotide sequence encodingsaid Vpr polypeptide comprises a sequence having at least 90% sequenceidentity to at least 20 contiguous base pairs of SEQ ID NO:59; (j) anHIV Vpu polypeptide, wherein the polynucleotide sequence encoding saidVpu polypeptide comprises a sequence having at least 90% sequenceidentity to at least 20 contiguous base pairs of SEQ ID NO:60; and (k)an HIV Env polypeptide, wherein the polynucleotide sequence encodingsaid Env polypeptide comprises a sequence having at least 90% sequenceidentity to the full length of a sequence selected from the groupconsisting of SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, and SEQ IDNO:64.
 2. The nucleic acid of claim 1 which is a recombinant expressionsystem for use in a selected host cell, wherein the recombinantexpression system comprises an expression cassette of claim 1, whereinsaid polynucleotide sequence is operably linked to control elementscompatible with expression of the polynucleotide in the selected hostcell.
 3. The recombinant expression system of claim 2 wherein saidcontrol elements are selected from the group consisting of atranscription promoter, a transcription enhancer element, atranscription termination signal, polyadenylation sequences, sequencesfor optimization of initiation of translation, and translationtermination sequences.
 4. The recombinant expression system of claim 2wherein said transcription promoter is selected from the groupconsisting of CMV, CMV+intron A, SV40, RSV, HIV-Ltr, MMLV-ltr, andmetallothionein.
 5. An isolated cell comprising an expression cassetteof claim 1, wherein said polynucleotide sequence is operably linked tocontrol elements compatible with expression in the selected cell.
 6. Theisolated cell of claim 5 which is selected from the group consisting ofa mammalian cell, an insect cell, a bacterial cell, a yeast cell, aplant cell, an antigen presenting cell, a primary cell, an immortalizedcell, and a tumor-derived cell.
 7. A method for producing a polypeptideincluding HIV polypeptide sequences, comprising incubating isolatedcells of claim 5 under conditions for producing said polypeptide.
 8. Thenucleic acid of claim 1 which is a vector for use in generating animmune response in a mammalian subject, wherein the vector comprises anexpression cassette of claim 1, and wherein said polynucleotide sequenceis operably linked to control elements compatible with expression of thepolynucleotide in the subject.
 9. A method of DNA immunization of asubject, comprising, introducing a vector of claim 8 into said subjectunder conditions that are compatible with expression of said expressioncassette in said subject.
 10. The method of claim 9, wherein said vectoris: (a) a nonviral vector; (b) encapsulated in a liposome preparation;or (c) a viral vector.
 11. The method of claim 9 wherein said vector isdelivered using a particulate carrier.
 12. The method of claim 11wherein said vector is coated on a gold or tungsten particle and saidcoated particle is delivered to said subject using a gene gun.
 13. Themethod of claim 9 wherein said vector is a viral vector and the viralvector is selected from the group consisting of a retroviral vector, analphaviral vector, and a lentiviral vector.
 14. The method of claim 9wherein said subject is a mammal.
 15. The method of claim 14 whereinsaid mammal is a human.
 16. The method of claim 9 wherein cells of thesubject are transfected with the vector of claim 10 under conditionsthat permit the expression of said polynucleotide and production of saidpolypeptide, thereby eliciting an immunological response to saidpolypeptide.
 17. The method of claim 16 wherein said transfecting isdone ex vivo and said transfected cells are reintroduced into saidsubject.
 18. The method of claim 16 wherein said transfecting is done invivo in said subject.
 19. The method of claim 16 where said immuneresponse is a humoral immune response.
 20. The method of claim 16 wheresaid immune response is a cellular immune response.
 21. The method ofclaim 16 wherein the vector is administered intramuscularly,intramucosally, intranasally, subcutaneously, intradermally,transdermally, intravaginally, intrarectally, orally or intravenously.